Ion separation instrument

ABSTRACT

An ion separation instrument includes an ion source coupled to at least a first ion mobility spectrometer having an ion outlet coupled to a mass spectrometer. In one embodiment, the ion source includes a molecule separation instrument operable to separate ions in time according to a molecular characteristic such as ion retention time. The resultant instrument is thus operable to provide molecular information separated in time as functions of retention time, ion mobility and ion mass/charge. In another embodiment, the ion separation instrument includes first and second ion mobility instruments disposed in a cascade arrangement between the ion source and mass spectrometer, wherein the two ion mobility instruments are operable to separate ions in time each according to different ion mobility functions. For example, the two ion mobility instruments may have different flight tube lengths, operate at different temperatures, operate in the presence of different electric fields and/or operate in the presence of different gases. The resultant instrument is thus operable to provide molecular information separated in time according to at least two different functions of ion mobility as well as ion mass/charge.

CROSS-REFERENCE TO RELATED U.S. APPLICATION

[0001] This is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 09/313,492, filed May 17, 1999 and entitled IONMOBILITY AND MASS SPECTROMETER, which is a continuation-in-part of U.S.Pat. No. 5,905,258 entitled HYBRID ION MOBILITY AND MASS SPECTROMETER.

FIELD OF THE INVENTION

[0002] The present invention relates generally to instrumentation forcharacterization of molecules based at least on their structures andmass-to-charge ratios as gas-phase ions, and more specifically to suchinstrumentation which provides for rapid and sensitive analysis ofcomposition, sequence, and/or structural information relating to organicmolecules, including biomolecules, and inorganic molecules.

BACKGROUND OF THE INVENTION

[0003] Biological molecules, such as DNA, RNA, proteins, carbohydratesand glycoconjugates, are comprised of repeating subunits typicallyreferred to as residues. The sequence of such residues ultimatelydefines the structure and function of the biomolecule and determines howit will interact with other molecules.

[0004] A central part of almost all conventional sequencing strategiesis the analysis of complex sets of sequence-related molecular fragmentsby chromatography or by polyacrylamide gel electrophoresis (PAGE).PAGE-based automated sequencing instruments currently exist andtypically require a number of fluorescent dyes to be incorporated intothe base-specifically terminated biomolecule product, which is thenprocessed through the polyacrylamide gel. The discrete-length productmolecules are detected near the bottom of the gel by their emittedfluorescence following excitation by a radiation source.

[0005] Such automated instruments are typically capable of generatingsequence information for biomolecules having 500 or more residues at arate of 10-20 times faster than manual methods. However, both the manualand automated PAGE techniques suffer from several drawbacks. Forexample, both approaches are labor-intensive since a gel must beprepared for each sequencing run. Also, while automated PAGE systems mayoffer faster analysis times than a manual approach, the accuracy of suchsystems is limited by artifacts generated by non-uniform gel matricesand other factors. Such automated systems are generally not equipped toaccurately process the effects of such artifacts, which are typicallymanifested as “smiling” compressions, faint ghost bands, and the like.Manual interpretation of such results is therefore often required whichsignificantly increases analysis time.

[0006] Researchers have, within the past several years, recognized aneed for more rapid and sensitive techniques for analyzing the structureand sequences of biomolecules. Mass spectrometry (MS) techniques, suchas time-of-flight mass spectrometry (TOFMS) and Fourier Transformion-cyclotron-resonance mass spectroscopy, are well known techniques forquickly and accurately providing ion mass information from whichsequence and structural determinations can be made. As is known in theart, TOFMS systems accelerate ions, via an electric field, toward afield-free flight tube which terminates at an ion detector. Inaccordance with known TOFMS principles, ion flight time is a function ofion mass so that ions having less mass arrive at the detector morequickly than those having greater mass. Ion mass can thus be computedfrom ion flight time through the instrument. FIG. 1 demonstrates thisprinciple for a cytochrome-c sample, having a known mass to charge ratio(m/z) of 12,360 da, and a lysozyme sample, having a known mass to chargeratio (m/z) of 14,306 da. In FIG. 1, signal peak 10, having a flighttime of approximately 40.52 μs corresponds to the lighter cytochrome-csample, and signal peak 12, having a flight time of approximately 41.04μs, corresponds to the heavier lysozyme sample.

[0007] Due to the significantly decreased sample preparation andanalysis times of MS techniques over the above-described PAGE technique,several MS sequencing strategies have recently been developed. Such MSsequencing techniques are generally operable to measure the change inmass of a biomolecule as residues are sequentially removed from its end.Examples of two such techniques, each involving elaborate pre-MSprocessing techniques, are described in U.S. Pat. Nos. 5,210,412 toLevis et al. and 5,622,824 to Köster.

[0008] In order to provide for the capability of determining sequenceand structural information for large biomolecules, it has beenrecognized that MS techniques must accordingly be capable of generatinglarge ions. Currently, at least two techniques are known for generatinglarge ions for spectral analysis; namely electrospray ionization (ESI)and matrix assisted laser desorption ionization (MALDI). While bothlarge ion generating techniques are readily available, known MStechniques are limited in both the quantity and quality of discernableinformation. Specifically, for large biomolecules, defined here as thosecontaining at least 50 residues, mass spectra of parent and sequencerelated fragment ions become congested to the degree that mass (TOF)peaks overlap.

[0009] One solution to the problem of congested mass spectra is toincrease the mass resolution capability of the MS instrument. Recentefforts at increasing such resolution have been successful, and completesequence information for a 50 base pair DNA has been obtained using aFourier Transform ion cyclotron resonance (FTICR) instrument. However,such instruments are extremely expensive, not readily available, andbecause of their extremely high vacuum requirements, they are generallynot suitable for routinely sequencing large numbers of samples.

[0010] Another solution to the problem of congested mass spectra is topre-separate the bulk of ions in time prior to supplying them to the ionacceleration region of the MS instrument. Mass spectrometry can then beperformed sequentially on “packets” of separated ion samples, ratherthan simultaneously on the bulk of the generated ions. In this manner,mass spectral information provided by the MS instrument may be spreadout over time in a dimension other than mass to thereby reduce thelocalized congestion of mass information associated with the bulk ionanalysis.

[0011] One known ion separation technique which may be used topre-separate the bulk of the ions in time prior to MS analysis is ionmobility spectrometry (IMS). As is known in the art, IMS instrumentstypically include a pressurized static buffer gas contained in a drifttube which defines a constant electric field from one end of the tube tothe other. Gaseous ions entering the constant electric field area areaccelerated thereby and experience repeated collisions with the buffergas molecules as they travel through the drift tube. As a result of therepeated accelerations and collisions, each of the gaseous ions achievesa constant velocity through the drift tube. The ratio of ion velocity tothe magnitude of the electric field defines an ion mobility, wherein themobility of any given ion through a high pressure buffer gas is afunction of the collision cross-section of the ion with the buffer gasand the charge of the ion. Generally, compact conformers, i.e. thosehaving smaller collision cross-sectional areas, have higher mobilities,and hence higher velocities through the buffer gas, than diffuseconformers of the same mass, i.e. those having larger collisioncross-sectional areas. Thus, ions having larger collision cross-sectionsmove more slowly through the drift tube of an IMS instrument than thosehaving smaller collision cross-sections, even though the ions havingsmaller collision cross-sections may have greater mass than those havinghigher collision cross-sections. This concept is illustrated in FIG. 2which shows drift times through a conventional IMS instrument for threeions, each having a different mass and shape (collision cross-section).As is evident from FIG. 2, the most compact ion 14 (which appears tohave the greatest mass) has the shortest drift time peak 16 ofapproximately 5.0 ms, the most diffuse ion 18 has the longest drift timepeak 20 of approximately 7.4 ms, and the ion 22 having a collisioncross-section between that of ion 14 and ion 18 (which also appears tohave the least mass), has a drift time peak 24 of approximately 6.1 ms.

[0012] Referring now to FIG. 3, an ion time-of-flight spectrum 26,obtained from a known time-of-flight mass spectrometer, is shown plottedvs. ion drift time. In-this figure, ions of different-mass are dispersedover different times of flight in the mass spectrometer. However, due tothe limited resolution of the mass spectrometer, ions are not completelyseparated in the spectrum, i.e. dots corresponding to different ionsoverlap. When compared with FIG. 6, which will be discussed more fullyin the DESCRIPTION OF THE PREFERRED EMBODIMENTS section, it is evidentthat different ions can be better resolved by an instrument thatseparates ions in two dimensions, namely ion mobility and ion mass.

[0013] Guevremont et al. have recently modified an existing IMS/MSinstrument to convert a quadrupole MS to a TOFMS [R. Guevremont, K. W.M. Siu, and L. Ding, PROCEEDINGS OF THE 44^(TH) ASMS CONFERENCE, (1996),Abstract]. Ions are generated in the Guevremont et al. instrument viaelectrospray, and 5 ms packets are gated into the IMS instrument. Theion packets produced by the IMS instrument are passed through a smallopening into an ion acceleration region of the TOFMS.

[0014] While Guevremont et al. have had some experimental success incoupling an IMS instrument to a TOFMS instrument, their resultinginstrumentation and techniques have several drawbacks associatedtherewith. For example, since the Guevremont et al. abstract discussesusing 5 ms gate pulses to admit ions into the IMS instrument, it isnoted that the resultant IMS spectrum has low resolution with at least 5ms peak widths. Secondly, because the drift tube and ion flight tube ofthe Guevremont et al. instrument are colinear, any spatial and temporalspread in an ion packet leaving the IMS leads directly to a spatial andtemporal spread of ions in the ion acceleration region of the TOFMS.These two characteristics lead to poor mass resolution in the TOFMS. Thecombination of low resolution in the IMS and low resolution in the TOFMSmakes this instrument incapable of resolving complex mixtures. What istherefore needed is a hybrid IMS/TOFMS instrument optimized to resolvecomplex mixtures. Such an instrument should ideally provide foroptimization of the ion mobility spectrum as well as optimization of themass spectrum. Moreover, such a system should provide for an optimuminterface between the two instruments to thereby maximize thecapabilities of the TOFMS.

SUMMARY OF THE INVENTION

[0015] The foregoing drawbacks associated with the prior art systemsdiscussed in the BACKGROUND section are addressed by the presentinvention. In accordance with one aspect of the present invention, amethod of separating ions in time comprises the steps of separating abulk of ions in time as a function of a first molecular characteristic,sequentially separating in time as a function of ion mobility at leastsome of the ions previously separated in time as a function of a firstmolecular characteristic, and sequentially separating in time as afunction of ion mass at least some of the ions previously separated intime as a function of ion mobility.

[0016] In accordance with another aspect of the present invention, anapparatus for separating ions in time comprises means for separating abulk of ions in time as a function of a first molecular characteristic,an ion mobility spectrometer (IMS) having an ion inlet coupled to themeans for separating a bulk of ions in time as a function of a firstmolecular characteristic and an ion outlet, wherein the IMS is operableto separate ions in time as a function of ion mobility. A massspectrometer (MS) is further included and has an ion acceleration regioncoupled to the ion outlet of the IMS, wherein the MS is operable toseparate ions in time as a function of ion mass.

[0017] In accordance with a further aspect of the present invention, amethod of separating ions in time comprises the steps of separating abulk of ions in time according to a first function of ion mobility,sequentially separating in time according to a second function of ionmobility at least some of the ions separated in time according to thefirst function of ion mobility, wherein the second function of ionmobility is different from the first function of mobility, andsequentially separating in time as a function of ion mass at least someof the ions separated in time according to the second function of ionmobility.

[0018] In accordance with still another aspect of the present invention,an apparatus for separating ions in time comprises a first ion mobilityspectrometer (IMS1) having an ion inlet and an ion outlet, wherein theIMS1 is operable to separate ions in time according to a first functionof ion mobility and a second ion mobility spectrometer (IMS2) having anion inlet coupled to the ion outlet of the IMS1 and an ion outlet,wherein the IMS2 is operable to separate ions in time according to asecond function of ion mobility different from the first function of ionmobility. A mass spectrometer is also included and has an ionacceleration region coupled to the ion outlet of the IMS2, wherein themass spectrometer is operable to separate ions in time as a function ofion mass.

[0019] One object of the present invention is to provide instrumentationfor rapid analysis and sequencing of large biomolecules, as well asanalysis of mixtures of organic and inorganic molecules.

[0020] Another object of the present invention is to provide an ionmobility and mass spectrometer for composition, sequence and structuralanalysis of biomolecules.

[0021] Yet another object of the present invention is to provide such aninstrument operable to produce molecular information separated in timeaccording to at least three different molecular characteristicfunctions.

[0022] Still another object of the present invention is to provide suchan instrument wherein two of the three different molecularcharacteristic functions are ion mobility and ion mass/charge, andwherein the third molecular characteristic function may be ion retentiontime, a second different ion mobility or the like.

[0023] Still a further object of the present invention is to provide atechnique for operating such an instrument in obtaining sequencinginformation.

[0024] These and other objects of the present invention will become moreapparent from the following description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 is a MALDI-TOF mass spectrum of cytochrome-c and lysozyme.

[0026]FIG. 2 is an IMS drift time distribution for three ions havingdifferent collision cross-sections.

[0027]FIG. 3 is a mass spectrum plotted against drift time illustratingthe limited resolution of a time-of-flight mass spectrometer.

[0028]FIG. 4 is a cross-section and schematic diagram of one embodimentof a hybrid ion mobility and time-of-flight mass spectrometer, inaccordance with the present invention.

[0029]FIG. 5 is a cross-section and schematic diagram of an alternateembodiment of a hybrid ion mobility and time-of-flight massspectrometer, according to the present invention.

[0030]FIG. 6 is a plot of ion time-of-flight vs. ion drift time foroligothymidine, utilizing the hybrid instrumentation of either FIG. 4 orFIG. 5.

[0031]FIG. 7A is a diagrammatic illustration of one preferred embodimentof an ion source for use with any of the instrument configurations shownin FIGS. 4, 5 and 9.

[0032]FIG. 7B is a diagrammatic illustration of an alternate embodimentof an ion source for use with any of the instrument configurations shownin FIGS. 4, 5 and 9.

[0033]FIG. 7C is a diagrammatic illustration of another alternateembodiment of an ion source for use with any of the instrumentconfigurations shown in FIGS. 4, 5 and 9.

[0034]FIG. 8A is a plot of ion intensity vs. ion drift time for an IMSinstrument without an ion trap disposed between the ion source and theIMS instrument.

[0035]FIG. 8B is a plot of ion intensity vs. ion drift time for an IMSinstrument having an ion trap disposed between the ion source and theIMS instrument.

[0036]FIG. 9 is a block diagram illustration of another alternateembodiment of an ion mobility and time-of-flight mass spectrometer, inaccordance with the present invention.

[0037]FIG. 10 is a partial cross-sectional diagram of yet anotheralternate embodiment of an ion source for use with any of the instrumentconfigurations shown in FIGS. 4, 5 and 9.

[0038]FIG. 11 is a cross-section of one preferred embodiment of thequadrupole mass filter illustrated in FIG. 9 as viewed along sectionlines 11-11.

[0039]FIG. 12 is a plot of ion intensity vs. mass-to-charge ratioillustrating operation of the quadrupole mass filter of FIG. 11.

[0040]FIG. 13 is a flowchart illustrating one preferred embodiment of aprocess for conducting sequencing analysis using the instrumentconfiguration of FIG. 9, in accordance with the present invention.

[0041]FIG. 14 is composed of FIGS. 14A-14D and illustrates an exampleion mass/mobility spectrum resulting from a first pass through theprocess illustrated in FIG. 13.

[0042]FIG. 15 is composed of FIGS. 15A-15D and illustrates an exampleion mass/mobility spectrum resulting from a second pass through theprocess illustrated in FIG. 13.

[0043]FIG. 16 is composed of FIGS. 16A-16D and illustrates an exampleion mass/mobility spectrum resulting from a third pass through theprocess illustrated in FIG. 13.

[0044]FIG. 17 is a block diagram illustrating alternative structuralvariations of the ion mobility and time-of-flight mass spectrometer ofthe present invention.

[0045]FIG. 18 is a block diagram illustrating further alternativestructural variations of the ion mobility and time-of-flight massspectrometer of the present invention.

[0046]FIG. 19 is a block diagram illustration of yet another alternateembodiment of an ion mobility and time-of-flight mass spectrometer, inaccordance with the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] For the purposes of promoting an understanding of the principlesof the invention, reference will now be made to the embodimentsillustrated in the drawings and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of the invention is thereby intended, such alterations andfurther modifications in the illustrated devices, and such furtherapplications of the principles of the invention as illustrated thereinbeing contemplated as would normally occur to one skilled in the art towhich the invention relates.

[0048] Referring now to FIG. 4, one preferred embodiment of a hybrid ionmobility and time-of-flight mass spectrometer instrument 30, inaccordance with the present invention, is shown. Instrument 30 includes,as its basic components, an ion source region 32 in communication withan ion mobility spectrometer 34, which itself is in communication with amass spectrometer 36. A computer-38 is provided for controlling at leastsome portions of the instrument 30 as well as for collecting ioninformation from mass spectrometer 36. Computer 38 is preferably apersonal computer (PC) of known construction having at least a known 386processor, although the present invention contemplates that computer 38may be any known computer, controller or data processor capable ofcontrolling instrument 30, as set forth in greater detail hereinafter,and of collecting and processing ion information from mass spectrometer36.

[0049] Preferably, mass spectrometer 36 is of the linear time-of-flighttype, although the present invention contemplates that spectrometer 36may alternatively be a known reflectron time-of-flight massspectrometer, multi-pass time-of-flight mass spectrometer, FourierTransform ion-cyclotron-resonance (FTICR-MS) mass spectrometer or otherknown mass spectrometer. Throughout this description, any massspectrometer will typically be referred to as a time-of-flight massspectrometer (TOFMS), although it is to be understood that any of theforegoing mass spectrometer instruments may be substituted thereforewithout detracting from the scope of the present invention. In any case,TOFMS 36 is, in one preferred embodiment, configured to maximize massresolution by minimizing the deleterious effects of initial ion positionand initial ion velocity distributions. Details of such a TOFMSconfiguration and operation thereof are given in U.S. Pat. Nos.5,504,326, 5,510,613 and 5,712,479 to Reilly et al., all assigned to theassignee of the present invention, and the contents of which are allincorporated herein by reference.

[0050] Ion mobility spectrometer (IMS) 34 includes a drift tube 40having a gas port 42 disposed adjacent to an ion exit end 44 of tube 40,wherein port 42 is connected to a source of buffer gas 46. The flow rateof buffer gas may be controlled by computer 38 via signal path 48, ormay alternatively be controlled by a manually actuated valve (notshown). Ion exit end 44 of drift tube 40 includes an endplate 43attached thereto, wherein endplate 43 defines an opening, or ionaperture, 45 therethrough.

[0051] Drift tube 40 includes a number of guard rings 50 distributedalong its inner surface, wherein the guard rings 50 are interconnectedby equivalent-valued resistors (not shown). The guard ring positionedmost adjacent to ion source region 32 is connected to a voltage sourceVS1 52 via signal path 54, and source 52 is preferably controlled bycomputer 38 via signal path 56, although the present inventioncontemplates controlling source 52 via a manual actuator (not shown).The drift tube 40 defines a longitudinal axis 72 therethrough which willbe referred to hereinafter as the drift tube axis 72. Voltage source 52is preferably set to a positive voltage to thereby establish a constantelectric field directed along axis 72 in a direction indicated by arrow55. Those skilled in the art will recognize that the importance of theguard ring and voltage source arrangement of the spectrometer 34 liesnot in its specific structure, but in its ability to establish, asaccurately as possible, a constant electric field in the direction ofarrow 55. In this sense, the present invention contemplates that anyknown structure or arrangement may be used to establish such an electricfield within drift tube 40 in the direction of arrow 55. It is to beunderstood, however, that a constant electric field in the direction ofarrow 55 is established to accelerate positively charged ions towardtube end 44, and that such an electric field may be reversed to therebyaccelerate negatively charged ions toward tube end 44.

[0052] Drift tube 40 may optionally be surrounded by a variabletemperature housing 58 which is connected to a variable temperaturesource 60 via path 62, all of which are shown in phantom. In oneembodiment, variable temperature source 60 is a fluid holding tank andpath 62 is a conduit leading to housing 58 which, in this case, ispreferably sealed. A return conduit (not shown) is also connected to thefluid holding tank so that fluid from within the tank may be circulatedthrough housing 58. The fluid within the fluid holding tank may be aheated or cooled gas or liquid such as, for example, liquid nitrogen. Inan alternate embodiment, variable temperature source 60 is a knownelectrically actuatable temperature controller, and path 62 comprises apair of electrical conductors connected between the controller andhousing 58. In operation, temperature controller 60 is operable to heator cool housing 58 as desired. Regardless of the particular embodimentof housing 58, source 60 and path 62, the present invention contemplatesthat source 60 may furthermore be controlled by computer 38 via signalpath 64.

[0053] Drift tube 40 is further surrounded by a housing 70 which definesa tube end 66 covering an ion entrance end thereof, wherein tube end 66defines an opening, or ion aperture, 68 therethrough, and an ion exitopening, or aperture, 84 adjacent to endplate 43. Preferably, ion optics47 are positioned between openings 45 and 84 to focus ions exitingopening 45 into an ion acceleration region of TOFMS 36. Openings 45, 68and 84 are preferably bisected by drift tube axis 72. An ion source 74,which will be described more fully hereinafter, is positioned within ionsource region 32 and is operable, preferably under the control ofcomputer 38 via a number, N, of signal paths 76, wherein N may be anypositive integer, to direct ions within the spectrometer 34 via opening68. Ions entering drift tube 40 separate in time as a function of theirindividual mobilities, as discussed hereinabove, and are sequentiallydirected through opening 70 toward TOFMS 36.

[0054] Housing 70 includes a pump 80 for controlling the pressure of thebuffer gas. Preferably, pump 80 is a diffusion pump, the operation ofwhich may be controlled by computer 38 via signal path 82.Alternatively, pump 80 may be manually controlled by a manual pumpactuator (not shown). In any case, pump 80 is operable to establish adesired pressure of the static buffer gas within drift tube 40. Inaccordance with known IMS techniques, the buffer gas within drift tube40 may typically be set within the range of between approximately oneand a few thousand Torr.

[0055] TOFMS 36 is preferably surrounded by a housing 126 that isattached to IMS 34. TOFMS 36 includes a first electrically conductivegrid or plate 86 connected to a second voltage source VS2 88 via signalpath 90, which is preferably controlled by computer 38 via signal path92. A second electrically conductive grid or plate 94 is connected to athird voltage source VS3 96 via signal path 98, which is preferablycontrolled by computer 38 via signal path 100. A third electricallyconductive grid or plate 102 is connected to a fourth voltage source VS4via signal path 106, which is preferably controlled by computer 38 viasignal path 108. Grids or plates 86, 94 and 102 define first and secondion acceleration regions therebetween as is known in the art, and whichwill be more fully described hereinafter. Those skilled in the art willrecognize that other known ion acceleration region structures may beused with TOFMS 36, such as, for example, positioning a fourth grid orplate between grids or plates 94 and 102.

[0056] Grid or plate 102 has a plate surface attached to one end of aflight tube 110, the opposite end of which is attached to a surface of afourth electrically conductive grid or plate 112. An ion detector 116 isdisposed adjacent to grid or plate 112 with an air gap 114 definedtherebetween. Ion detector 116 is connected to a fifth voltage sourceVS5 118 via signal path 120, which is preferably controlled by computer38 via signal path 122. Ion detector 116 further has a signal outputconnected to computer 38 via signal path 124, whereby detector 116 isoperable to provide ion arrival time information to computer 38. Gridsor plates 86, 94, 102 and 112 are preferably arranged in juxtapositionwith each other such that all plate surfaces having greatest surfacearea are parallel with each other as well as to the surface of the iondetector 116, and are further preferably perpendicular to a longitudinalaxis 128 defined centrally through the flight tube 110, which willhereinafter be referred to as the flight tube axis 128.

[0057] TOFMS 36 further includes a pump 130 for controlling the vacuumof the TOFMS chamber defined by housing 126. Preferably, pump 130 is adiffusion pump, the operation of which may be controlled by computer 38via signal path 132. Alternatively, pump 130 may be manually controlledby a manual pump actuator (not shown). In any case, pump 130 is operableto establish a desired vacuum within housing 126 which may be set, inaccordance with know TOFMS operating techniques, to within the range ofbetween approximately 10⁻⁴ and 10⁻¹⁰ Torr.

[0058] In the instrument 30 illustrated in FIG. 4, TOFMS 36 ispreferably arranged relative to IMS 34 such that the flight tube axis128 is perpendicular to the drift tube axis 72. Moreover, TOFMS 36 ispreferably positioned relative to IMS 34 such that the drift tube axis72 and the flight tube axis 128 bisect within the first ion accelerationregion defined between grids or plates j86 and 94. In an alternativeconfiguration of TOFMS 36, grid or plate 94 may be omitted, and theTOFMS 36 need then be positioned relative to IMS 34 such that the drifttube axis 72 bisects the flight tube axis 128 within the ionacceleration region defined between grids or plates 86 and 102. Ineither case, TOFMS is preferably positioned relative to IMS 34 such thatthe drift tube axis 72 bisects the flight tube axis 128 approximatelycentrally within the region of interest.

[0059] In the operation of instrument 30, ions are generated by ionsource 74, in accordance with one or more ion generation techniquesdescribed hereinafter, and are supplied to IMS 34 via IMS inlet opening68. A buffer gas typically used in IMS instruments 34 is supplied todrift tube 40 via buffer gas source 46, wherein the buffer gas isregulated to a desired pressure via pump 80, buffer gas source 46 or acombination thereof. Typically, the buffer gas is regulated to apressure of between approximately 1 and a few thousand Torr. Voltagesource 52 supplies a voltage sufficient to generate a constant electricfield along the drift tube axis in a direction indicated by arrow 55.

[0060] In accordance with known IMS 34 operation, ions entering IMSinlet opening 68 travel through drift tube 40 toward IMS outlet opening84, wherein the ions separate in time according to their individualmobilities. Ions having low mobility lag behind those having highermobility, wherein ion mobilities are largely a function of theircollision cross-sections. As a result, the more compact ions arrive atthe IMS outlet opening 84 more quickly than more diffuse ions. Thoseskilled in the art will recognize that the temperature of drift tube 40may also be controlled via variable temperature source 60 so that ionmobility analysis may be performed as a function of temperature.

[0061] TOFMS 36 is operable to accelerate ions from the space definedbetween grids or plates 86 and 94 toward a field-free flight tube 110,wherein the ions separate in time according to their individual masses.Generally, ions having less mass will reach the detector 116 morequickly than those having greater mass. The detector 116 is operable todetect arrival times of the ions thereat and provide signalscorresponding thereto to computer 38 via signal path 124.

[0062] As set forth in greater detail in U.S. Pat. Nos. 5,504,326,5,510,613 and 5,712,479 to Reilly et al., which have been incorporatedherein by reference, voltage sources VS2 88, VS3 96 and VS4 104 aretypically controlled by computer 38 to initially establish voltages atgrids or plates 86, 94 and 102 that match the voltage level associatedwith IMS 34 (which is set by voltage source VS1 52). Depending uponvarious instrument parameters, such as the length of flight tube 110,the distances between grids or plates 88, 94, 102 and 112, and thedistance 114 between grid or plate, 112 and detector 116, as well asestimates of initial ion position or initial ion velocity within thespace defined between grids or plates 86 and 94, computer 38 is operableto control sources 88, 96 and/or 104 to instantaneously increase theelectric field between grids or plates 86, 94 and 102 to thereby createan ion drawout electric field therebetween which accelerates ionsbetween these grids toward flight tube 110. Preferably, the pulsed iondrawout electric field is in a direction from grid or plate 86 towardflight tube 110 to thereby accelerate positively charged ions toward theflight tube 110. Those skilled in the art will recognize, however, thatthis electric field may alternatively be reversed to acceleratenegatively charged ions toward the flight tube 110.

[0063] In any event, ions within the space defined between grids orplates 86 and 94 are accelerated by the pulsed ion drawout electricfield to the space defined between grids or plates 94 and 102. Due tothe fact that ions entering the region defined between grids or plates86 and 94 along axis 72 have a narrow spatial distribution, due tofocusing of the ions into this region via ion optics 47, and a smallvelocity component along axis 128, it is possible to choose the pulsedvoltage applied to grids or plates 86 and/or 94 in such a way as toobtain sharp TOFMS peaks. The goal of the pulsed ion drawout electricfield and the subsequent acceleration of the ions between grids orplates 94 and 102 is to provide all ions reaching grid or plate 102 withsubstantially the same kinetic energy. The flight tube 110 has noelectric field associated therewith so that the ions drift from grid orplate 102 toward detector 116, wherein the ions separate in time as afunction of their individual masses as described hereinabove. Computer38 typically controls voltage source VS5 118 to supply a voltage theretoduring detection times to thereby increase the gain of detector 116 asis known in the art. Pump 130 controls the vacuum within TOFMS 36, andpump 130 is preferably controlled by computer 38 via signal path 132.TOFMS 36 is typically operated between 10⁻⁴ and 10⁻¹⁰ Torr.

[0064] In the embodiment 30 of the hybrid IMS/TOFMS instrumentillustrated in FIG. 4, drift tube axis 72 preferably bisects the spacedefined between grids or plates 86 and 94 of TOFMS 36, and isperpendicular to flight tube axis 128. The present inventionalternatively contemplates arranging TOFMS 36 relative to IMS 34 suchthat the drift tube axis 72 passes between grids or plates 86 and 94perpendicular to flight tube axis 128, but at some other known distancerelative to either of the grids or plates 86 and 94. In either case, theforegoing structural positioning of TOFMS 36 relative to IMS 34 providesadvantages over non-perpendicular arrangements of the drift tube axis 72relative to the flight tube axis 128. For example, such a perpendiculararrangement ensures that ion packets entering the ion accelerationregion defined between grids or plates 86 and 94 from IMS 34 will haveconstant and relatively well defined initial ion positions as theytravel therebetween along axis 72. As discussed briefly hereinabove, ionoptics 47 focus ions into the ion acceleration region to therebyminimize spatial distribution of the ions. Moreover, since axis 72 isparallel with grids or plates 86 and o4, ion position with respect toaxis 128 will remain relatively constant. This feature provides for theability to accurately estimate initial ion position within the ionacceleration region defined between grids or plates 86 and 94, tothereby allow a more accurate estimation of the pulsed ion drawoutelectric field discussed above.

[0065] Preferably, computer 38 controls the generation of ions from ionsource 74, as will be discussed in greater detail hereinafter, so thatcomputer 38 has knowledge of the times at which ions were introducedinto IMS 34, hereinafter referred to as ion introduction events. Thecomputer 38 is then operable to control voltage sources 88 and 96 torepeatedly provide the pulsed ion drawout field some number of times forevery ion introduction event. In one embodiment, a pulsed ion drawoutfield is repeatedly provided 512 times for every ion introduction event.Those skilled in the art will recognize that the number of pulsed iondrawout fields provided for every ion introduction event is directlyproportional to the ultimate resolution of the instrument 30. As thispulsed operation relates to some of the advantages of the perpendicularpositioning of TOFMS 36 relative to IMS 34, such an arrangementminimizes the possibility that all or part of any one ion packet willtravel through the TOFMS 36 unprocessed. Due to the direction of travelof the ion packets relative to the grids or plates 86 and 94, and alsoto the pulsed nature of the ion drawout electric field, the TOFMS 36will have multiple chances to accelerate each ion packet toward detector116 as they travel along axis 72. As such, the instrument 30 isconfigured to provide for maximum ion throughput to detector 116.

[0066] Referring now to FIG. 5, an alternate embodiment of a hybrid ionmobility and time-of-flight mass spectrometer 150, in accordance withthe present invention, is shown. Spectrometer 150 is similar in manyrespects to spectrometer 30 shown in FIG. 4 and described hereinabove,and like components are therefore identified with like numbers.Discussion of the common components, as well as the basic operation ofIMS 34 and TOFMS 36′, will therefore not be repeated for brevity's sake.

[0067] Unlike instrument 30 of FIG. 4, the TOFMS 36′ of instrument 150is positioned relative to IMS 34 such that the drift tube axis 72 alsodefines the flight tube axis of TOFMS 36′. Alternatively, TOFMS 36′could be arranged relative to IMS 34 with any orientation such that thedrift tube axis 72 is non-perpendicular to the flight tube axis. In anysuch orientation, the initial positions of the ion packets within thespace defined between grids or plates 86′ and 94 either cannot beestimated with any degree of accuracy (as in the orientationillustrated) or changes as the ion packets travel along axis 72 (as inany non-perpendicular arrangement). Moreover, in any such orientation,it is difficult to estimate when, relative to an ion introduction event,the ion packets will arrive within the space defined between grids orplates 86′ and 94, and the timing of the pulsed ion drawout electricfields is thus difficult to predict. As a result, it is likely that thetiming of the pulsed ion drawout electric fields will be inaccurate sothat ions may be lost within the TOFMS 36′ and/or the mass resolution ofthe TOFMS 36′ u will be adversely affected.

[0068] In order to address the foregoing problems associated withnon-perpendicular positioning of the TOFMS 36′ relative to the IMS 34,which are the same problems associated with the Guevremont et al. systemdiscussed hereinabove in the BACKGROUND section, instrument 150 isprovided with an ion trap 152 operatively positioned between the ionoutlet opening 84 of IMS 34 and the space defined between grids orplates 86′ and 94. In the embodiment illustrated in FIG. 5, grid orplate 86′ defines an ion inlet opening 178 therethrough which is alignedalong axis 72 with ion outlet opening 84 of IMS 34. In othernon-perpendicular arrangements of TOFMS 36′ relative to IMS 34, ioninlet opening 178 may not be required since ions may enter the spacebetween grids or plates j86′ and 94 in the same manner as discussed withrespect to the embodiment 30 illustrated in FIG. 4.

[0069] In any event, ion trap 152 is preferably a known quadrupole iontrap having a first endcap 154, a center ring 162 and a second endcap170. Each of the endcaps 154 and 170 define apertures therethrough whichalign with axis 72. In this configuration, ion trap 152 confines ionstherein to a small volume in its center which is in alignment with theion inlet opening to TOFMS 36′. First endcap 154 is connected to avoltage source VS6 156 via signal path 158, which is itself connected tocomputer 38 via signal path 160. Center ring 162 is connected to avoltage source VS7 164 via signal path 166, which is itself connected tocomputer 38 via signal path 168, and second endcap 170 is connected to avoltage source VS8 172 via signal path 174, wherein source 172 isconnected to computer 38 via signal path 176. Preferably, sources 156and 172 are operable to produce DC voltages and source 164 is operableto produce AC voltages in the RF range.

[0070] In operation, computer 38 controls sources 156 and 172 to biasendcaps 154 and 170 such that ions exiting ion outlet opening 84 of IMS34 have just enough energy to enter the opening defined in the firstendcap 154. Once therein, the ions collide with buffer gas leaking outof opening 84 into the trap 152, and lose sufficient energy thereby sothat the RF voltage on center ring 162 is operable to confine the ionswithin the trap 152. The confined ions undergo further collisions insidethe trap 152 which causes the ions to correspondingly experience furtherenergy loss, resulting in a concentration of the ions toward the centerof ring 162 due to the RF voltage thereon. As long as the voltages onendcaps 154 and 170 and center ring 162 are maintained, ions may enterthe trap 152 and collect therein. Ions are ejected out of the trap 152by turning off the RF voltage on center ring 162 and applying anappropriate DC pulse to one of the endcaps 154 or 170. For example, toeject a collection of positively charged ions from trap 152, either thevoltage on endcap 154 may be pulsed above that present on endcap 170 orthe voltage on endcap 170 may be pulsed below that present on endcap154. In general, the magnitude of the RF field applied to the centerring via source 164, as well as any DC voltage included therein, may bevaried to thereby select ions of any desired mass to charge ratio to becollected by ion trap 152. Ions of all mass to charge ratios, or ions ofany particular mass to charge ratio, may be selectively collected withinion trap 152 through proper choice of DC level and RF peak magnitudeprovided by voltage source 164.

[0071] As it relates to the present invention, the ion trap 152 iscontrollable by computer 38 to periodically eject the collected ionpackets therefrom, hereinafter referred to as an ion ejection event, soas to provide for a more accurate estimate of initial ion positionwithin the space defined between grids or plates 86′ and 94. Since thecomputer 38 controls the time at which a packet of collected ions isejected from ion trap 152, the time at which the ion packet arrives at aspecified position in the space defined between grids or plates 86′ and94 can be accurately estimated. Knowing the approximate time, relativeto the ion ejection event, at which the ion packet arrives at thespecified position between grids or plates 86′ and 94, computer 38 maymore accurately estimate appropriate timing for applications of thepulsed ion drawout electric field to thereby provide for maximum massresolution as discussed hereinabove. Moreover, providing for a moreaccurate estimate of the timing of the pulsed ion drawout electricfields reduces the likelihood that ion packets, or at least portionsthereof, will be lost within the TOFMS 36′.

[0072] In the operation of instrument 150, IMS 34 is operable to providepackets of ions, which are separated in time as a function of ionmobility, to TOFMS 36′ via ion outlet opening 84. Computer 38 controlsion trap 152 to collect the various ion packets therein one at a time,and eject each collected ion packet therefrom at periodic intervals. Theejected ions enter the space defined between grids or plates 86′ and 94as discussed hereinabove, and computer 38 is operable to computerappropriate times at which to apply the pulsed ion drawout electricfields based on the timing of the ion ejection events. The TOFMS 36′ isthereafter operable as described hereinabove to produce mass spectruminformation.

[0073] Referring now to FIG. 6, a plot 190 of ion flight time vs. iondrift time for an oligothymidine sample is shown, wherein the data shownis producible via either instrument embodiment 30 or 150. As compared tothe plot of FIG. 3, it is apparent that the hybrid ion mobility andtime-of-flight mass spectrometer of the present invention is operable toresolve structural information of molecules in two substantiallyorthogonal dimensions. For each drift time, corresponding to arrival inthe TOFMS of a corresponding ion packet, the instrument of the presentinvention is operable to resolve a number of times-of-flight,corresponding to a number of mass to charge ratios. The plot 190 of FIG.6 thus illustrates that the total resolving power of instrument 30 isdrastically better than that achievable via an IMS or TOFMS alone. Thistechnique dramatically reduces the problem of congestion of massspectra, due to mass peak overlap, in obtaining sequence information forlarge biomolecules (in excess of 50 residues). The present inventionthus provides an instrument for composition, sequence and structuralanalysis of biomolecules which does not suffer from drawbacks associatedwith prior art systems discussed in the BACKGROUND section.

[0074] Referring now to FIG. 7A, one preferred embodiment 74′ of an ionsource 74 for either of the instrument embodiments of FIGS. 4 and 5, isshown. Embodiment 74′ includes a chamber 200 having a sample 202 mountedtherein and an optical window 206 extending therefrom. A radiationsource 204 is electrically connected to computer 38 via signal path 76A,and is configured to direct radiation through optical window 206 tothereby irradiate sample 202. Chamber 200 may include a conduitextending therefrom to a pump 208 which may be controlled by computer 38via signal path 76B.

[0075] Ion source 74′ is a known MALDI arrangement wherein radiationsource 204, preferably a laser, is operable to desorb gaseous ions froma surface of the sample 202. Computer 38 is operable to controlactivation times of laser 204 to thereby control sample ionizationevents. The desorbed ions are directed by the internal structure ofchamber 202 to ion inlet opening 68 of IMS 34. The sample 202 may, inaccordance with the present invention, be a biomolecule of any size suchas DNA, RNA, any of various proteins, carbohydrates, glycoconjugates,and the like. Pump 208 may be controlled to pressurize chamber 208 tothereby conduct high pressure MALDI analysis as is known in the art.

[0076] Referring now to FIG. 7B, an alternate embodiment 74″ of an ionsource 74 for either of the instrument embodiments of FIGS. 4 and 5, isshown. Embodiment 74″ includes a liquefied sample 220 having a sprayhose or nozzle 222 extending toward an opening defined in a desolvationregion 226. Actuation of the spray nozzle 222 may be manuallycontrolled, as is known in the art, or may be controlled by computer 38via signal path 76C. Desolvation region 226 is connected to computer 38via signal path 76C′, and is operable to convert charged sample dropletssupplied thereto via nozzle 222 into gaseous ions and supply these ionsto a ion optics member 228. Optics member 230 is operable to focus thegaseous ions and direct them into ion inlet opening of IMS 34. Ionsource region 32 includes a conduit extending therefrom to a pump 232which may be controlled by computer 38 via signal path 76D.

[0077] Ion source 74″ is a known electrospray ionization (ESI)arrangement operable to convert a liquefied solution containing thesample to gaseous ions. Computer 38 is operable to control activationtimes of desolvation region 226 to thereby control sample ionizationevents. Pump 232 is operable to pressurize the ion source region 32 asis known in the art, and the desolvation region 226 is operable convertthe liquefied solution to gaseous ions. The sample source 220 may, inaccordance with the present invention, include a solution containing abiomolecule of any size such as DNA, RNA, any of various proteins,carbohydrates, glycoconjugates, and the like.

[0078] Referring now to FIG. 7C, another alternate embodiment 74′″ of anion source 74 for either of the instrument embodiments of FIGS. 4 and 5,is shown. Embodiment 74′″ includes a sample source 236, which may beeither of the foregoing sample sources 74′ or 74″ illustrated in FIGS.7A or 7B, and which may be controlled as described hereinabove bycomputer 38 via a number, M, of signal paths 76E, wherein M may be anyinteger less than N (see FIGS. 4 and 5).

[0079] Ion source 74′″ further includes an ion trap 152 positionedbetween ion source 236 and the ion inlet opening 68 of IMS 34. Ion trap152 is preferably a known quadrupole ion trap identical to that shown inFIG. 5 and described hereinabove. A detailed discussion of the operationof ion trap 152 therefore need not be repeated here. Endcap 154 isconnected to a voltage source VS9 238 via signal path 240, center ring162 is connected to a voltage source VS10 242 via signal path 244 andendcap 170 is connected to a voltage source VS11 246 via signal path248. VS9, VS10 and VS11 are each connected to computer 38 via signalpaths 76F, 76G and 76H, respectively. Computer 38 is operable to controlVS9, VS10 and VS11 identically as described with respect to VS6, VS7 andVS8, respectively, of FIG. 5.

[0080] In operation, computer 38 is operable to control ion trap 152, ina manner similar to that described hereinabove, to collect a bulk ofions therein and selectively eject the collected ions therefrom towardion inlet opening 68 of IMS 34. As is known in the art, the peakresolution of an ion mobility instrument, such IMS 34, is limited by thelength of the input pulse of ions into the instrument. Generally,mobility peaks cannot be resolved any better than the time length of theinput ion pulse. A drawback particularly associated with the use of ESIis that the input ion pulse width must typically be at least 50 μs inorder to produce enough ions for analysis. However, with the ion sourcearrangement 74′″ shown in FIG. 7C, computer 38 is operable to collect alarge number of ions within ion trap 152 prior to pulsing the ions intothe IMS 34. With a sufficient number of ions collected in ion trap 34,the only limitation on the ion input pulse length, and hence theresolution capability of IMS 34, is the time required to open and closeion trap 152. With existing ion traps, the ion input pulse lengths maybe reduced to less than 1.0 μs in duration.

[0081]FIGS. 8A and 8B show a comparison of ion mobility distributionsfor a maltotetraose sample, wherein the spectrum 250 of FIG. 8A wasproduced using an ESI source similar to that shown in FIG. 7B, with100,083 input pulses of 20 μs duration. The spectrum 252 of FIG. 8B wasproduced using the same ESI source as that used for FIG. 8A along withan ion trap, such as ion trap 152 shown in FIG. 7C, with 4003 pulses of1 μs duration. Compared to spectrum 250, spectrum 252 has a 4-5 timesincrease in signal strength, an increase in resolution by a factor ofapproximately 20 and an increase in signal-to-noise ratio by a factor ofapproximately 20 as well.

[0082] Referring again to FIG. 7C, ion trap 152 may be used with anyknown ion generation source to increase not only the resolution andsensitivity of IMS 34 along, but also the resolution and sensitivity ofeither hybrid instrument 30 or 150 of FIGS. 4 and 5.

[0083] It is to be understood that either embodiment of the hybrid ionmobility and time-of-flight mass spectrometer shown and described hereinis capable of operation in a number of different operational modes. Forexample, the structure and operation of the various embodiments of thepresent invention have been described herein according to a first modeof operation wherein ions of relatively low energy are generated andinjected into the hybrid instrument, from which structural informationrelating to the ions can be obtained.

[0084] In a second mode of operation, such ions could be injected intothe hybrid instrument at higher energies, wherein high energy collisionswith the buffer gas within the IMS 34 result in ion fragmentation. Insuch a case, the ion fragments, separated in time as a function of theirmobilities, would be supplied to the TOFMS portion of the instrument,wherein mass spectra information of the various fragments could beobtained for sequencing analysis. Alternatively, fragmentation of ionsfor such analysis may be accomplished via any of a number of other knowntechniques. Examples of such known alternative ion fragmentationtechniques include enzyme degradation fragmentation,photo-fragmentation, thermal dissociation such as by heating drift tube40 via control of variable temperature source 60, electron impactdissociation, surface induced dissociation, and blackbody infraredradiation induced dissociation.

[0085] In a third mode of operation, ions of only a particular masscould be processed by the hybrid instrument. One way of generating ionsof only a particular mass is to adjust the peak amplitude and/or DCvoltage of the center ring voltage source of an ion trap positionedprior to the IMS 34. By properly adjusting this voltage, ion trap 152may be configured to store therein only ions having a particular mass tocharge ratio. In this manner, the ion trap 152 is controlled to act asan ion filter. Another way of analyzing ions of only a particular massis to provide an ion trap 152 between the IMS 34 and TOFMS 36, andcontrolling the ion trap 152 as just discussed to filter out ions havingundesirable mass to charge ratios.

[0086] In a fourth mode of operation, high energy ions of only aparticular mass are introduced into the IMS 34. Therein, these ionsundergo fragmentation, and such fragments could then be furtherprocessed by the TOFMS 36 as discussed above.

[0087] Referring now to FIG. 9, one preferred embodiment of an ionmobility and mass spectrometer instrument 300 that is particularly wellsuited for conducting sequencing analysis in a manner similar to thatjust described hereinabove with respect to the second mode of operation,in accordance with the present invention, is shown. Several of thecomponents of instrument 300 are identical to those shown and describedwith respect to FIGS. 4 and 5, and some of the structural andoperational details thereof will accordingly be omitted here forbrevity. For example, instrument 300 includes an ion source 32operatively connected to an ion mobility spectrometer (IMS), wherein IMS34 includes a source of buffer gas 46 that is controllable via operationof a pump 80 as described hereinabove. Instrument 300 further includes amass spectrometer (MS) 36, preferably a time-of-flight mass spectrometer(TOFMS), that is configured to receive ions from IMS 34 as describedhereinabove. In this embodiment, however, the drift tube axis of IMS 34(not shown in FIG. 9) and the flight tube axis of TOFMS 36 (not shown inFIG. 9) may be arranged at any desired angle with respect to each other.It has been determined through experimentation that fornon-perpendicular configurations of IMS 34 relative to TOFMS 36 (i.e.,configurations other than that illustrated in FIG. 4), an ion trap 152(see FIG. 5) is not required as described hereinabove if the ionacceleration region (between grids 86, 94 and 102) of TOFMS 36 iscontinually activated or pulsed. In other words, ions need not becollected in an ion trap 152 for timing purposes if the ion accelerationregion of TOFMS 36 is continually pulsed in a free-running operationalmode. Accordingly, ion trap 152 may be omitted from any perpendicular ornon-perpendicular configurations of the IMS drift tube axis relative tothe TOFMS flight tube axis, although the present invention contemplatesthat such an ion trap 152 may optionally be used in such configurationsas desired, wherein trap 152 may be positioned adjacent to the entranceof TOFMS 36.

[0088] Instrument 300 further includes a computer 310 having a memory312. Computer 310 is preferably operable to control the flow rate ofbuffer gas #1 within buffer gas source 46 via signal path 48, and isfurther preferably operable to control pump 80 of IMS 34 via signal path82 and a vacuum pump 130 of TOFMS 36 via signal path 132, as describedhereinabove. Computer 310 is also operable to control ion source 32 viaa number, N, of signal paths 76, wherein N may be any integer, and isfurther operable to receive ion detection signals from TOFMS 36 viasignal path 124 and process such signals to produce two-dimensional ionspectra; e.g. ion mass vs. ion mobility, as described hereinabove.

[0089] Instrument 300 includes a number, J, of voltage sources 314 ₁-314_(J) connected to computer 310 via signal paths 316 ₁-316 _(J). Voltagesources 314 ₁-314 _(J) are operatively connected to IMS 34 viacorresponding signal paths 318 ₁-318 _(J). In operation, computer 310 isoperable to control voltage sources 314 ₁-314 _(J) to thereby controlthe operation of IMS 34 as described hereinabove. Instrument 300 furtherincludes another number, M, of voltage sources 330 ₁-330 _(M) connectedto computer 310 via signal paths 332 ₁-332 _(M). Voltage sources 330₁-330 _(M) are operatively connected to TOFMS 36 via correspondingsignal paths 334 ₁-334 _(M). In operation, computer 310 is operable tocontrol voltage sources 330 ₁-330 _(M) to thereby control the operationof TOFMS 36 as described hereinabove.

[0090] The components of instrument 300 described thus far with respectto FIG. 9 are identical to previously described components of theinstruments 30 and/or 150 of FIGS. 4 and 5. Unlike instruments 30 and150, however, instrument 300 further includes a quadrupole mass filter302 having an ion inlet coupled to the ion outlet of IMS 34 and an ionoutlet coupled to an ion inlet of a collision cell 304 of knownconstruction. An ion outlet of collision cell 304 is coupled to an ioninlet of TOFMS 36; i.e., to the ion acceleration region defined betweenplates or grids 86 and 94 of TOFMS as shown in FIGS. 4 and 5. Collisioncell 304 includes a source of buffer gas 306, wherein the flow rate ofbuffer gas #2 is controlled by computer 310 via signal path 307,preferably in a manner described hereinabove with respect to thecomputer control of the buffer gas source 46 of FIG. 4. Alternatively,buffer gas source 306 may be omitted and buffer gas source 46 may beconfigured to provide buffer gas #1 to cell 304 via conduit 305 as shownin phantom in FIG. 9. Collision cell 304 further includes a pump 308 ofknown construction, the operation of which is controlled by computer 310via signal path 309. As is known in the art, pump 308 may be controlledto establish and maintain a desired quantity of buffer gas withincollision cell 304, and may further be controlled to purge cell 304 ofbuffer gas. Alternatively, structure 308 may represent a manuallyactuatable or computer controlled valve. In this case, valve 308 may becontrolled to establish and maintain a desired quantity of buffer gas #2within collision cell 304, or may alternatively be controlled toestablish and maintain a desired quantity of buffer gas #1 within thequadrupole mass filter 302 and collision cell 304.

[0091] A number, K, of voltage sources 320 ₁-320 _(K) are provided,wherein K may be any integer, and wherein control inputs of sources 320₁-320 _(K) are connected to computer 310 via corresponding signal paths322 ₁-322 _(K). Outputs of voltage sources 320 ₁-320 _(K) areoperatively connected to the quadrupole mass filter (QMF) 302, in amanner to be described more fully hereinafter with respect to FIGS. 11and 12, via corresponding signal paths 324 ₁-324 _(K). A number, L, ofvoltage sources 326 ₁-326 _(L) are provided, wherein L may be anyinteger, and wherein control inputs of sources 326 ₁-326 _(L) areconnected to computer 310 via corresponding signal paths 328 ₁-328 _(L).Outputs of voltage sources 326 ₁-326 _(L) are operatively connected tothe collision cell 304 in a known manner via corresponding signal paths329 ₁-329 _(L).

[0092] Referring now to FIG. 10, a cross-section of another preferredstructure of the ion source 32 for use with any of the instrumentsillustrated in FIGS. 4, 5 and 9, in accordance with the presentinvention, is shown. Ion source 32 includes an ion source chamber 350separated from an ion collection chamber 354 by a wall or partition 355.Ion source chamber 350 includes a port having a conduit 352 connectedthereto, wherein conduit 352 is preferably connected to a pump or valveof known construction for changing gas pressure within region 350. Anion source 74 is disposed within region 350, wherein source 74 may beany of the ion sources 74′, 74″ or 74′″ described hereinabove withrespect to FIGS. 7A-7C, and/or any combination thereof. Wall orpartition 355 includes an aperture 353 therethrough that is aligned withan ion outlet of ion source 74 and is also preferably aligned with alongitudinal axis of the drift tube 40 of IMS 34, wherein aperture 353defines an ion inlet to ion collection chamber 354. An electricallyconductive grid, or series of vertically or horizontally parallel wires,356 (hereinafter “grid”) is positioned across the ion inlet aperture 68of IMS 34, wherein grid 356 is connected to one of the voltage sources314 ₁ via signal path 318 ₁. Computer 310 is operable to control thevoltage of grid 356, as is known in the art, to thereby permit andinhibit entrance of ions into IMS 34. For example, computer 310 isoperable to inhibit entrance of ions into IMS 34 by activating voltagesource 314 ₁ to thereby cause ions in the vicinity of grid 356 to beattracted thereto and neutralized upon contact. Conversely, computer 310is operable to permit entrance of ions into IMS 34 by deactivatingvoltage source 314 ₁ to thereby permit passage of ions therethrough.Alternatively, the ion gating function may be accomplished by a voltagesource 320 ₂ connected to guard rings 50 via signal path 318 ₂, whereincomputer 310 is operable to control source 320 ₂ to attract ions toguard rings 50 when it is desirable to inhibit ions from travelingthrough drift tube 40. In this case, grid 356 and voltage source 320 ₁may be omitted from FIG. 10. Alternatively still, the ion gatingfunction may be accomplished by impressing a voltage across aperture 68to thereby create an electric field therebetween. In this case, computer310 is operable to control the voltage across aperture 68 to divert ionstoward guard rings 50 when it is desirable to inhibit ions fromtraveling through drift tube 40. Those skilled in the art will recognizethat any known technique for pulsing ions from ion collection chamber354 through ion inlet aperture 68, including for example any knownelectrical, mechanical and/or electromechanical means, may be used, andthat any such technique falls within the scope of the present invention.

[0093] In any case, the ion collection chamber 354 is functionallysimilar to the ion trap 152 of FIG. 7C in that it provides for thecollection of a large quantity of ions generated by ion source 74 priorto entrance into IMS 34. Through appropriate control of ion source 74and grid 356 or equivalent, the quantity of ions entering IMS 34 maythus be correspondingly controlled.

[0094] Referring now to FIG. 11, a cross-section of the quadrupole massfilter (QMF) 302, as viewed along section lines 11-11 of FIG. 9, isshown. QMF 302 includes four electrically conductive rods or plates 360,362, 364 and 366 that are preferably disposed equidistant from alongitudinal axis 365 extending through QMF 302. Two of the opposingrods 360 and 362 are electrically connected to voltage source 320 ₁ viasignal path 324 ₁, wherein source 320 ₁ has a control input connected tocomputer 310 via signal path 322 ₁. Signal path 324 ₁ is connected to asignal phase shifter 366 of known construction via signal path 368,wherein a signal output of phase shifter 366 is electrically connectedto the remaining two opposing rods 364 and 366. Computer 310 is operableto control voltage supply 320 ₁, which is preferably a radio frequency(RF) voltage source, to thereby control the RF voltage applied to rods360 and 362. Phase shifter 366 is preferably operable to shift the phaseof the RF voltage on signal path 368 by 180° and apply this phaseshifted RF voltage to signal path 324 ₂. Those skilled in the art willrecognize that phase shifter 366 may alternatively be replaced with asecond RF voltage source that is controllable by computer 310 to producean RF voltage identical to that produced by source 320 ₁ except shiftedin phase by 180°. In any case, signal paths 324 ₁ and 324 ₂ areelectrically connected to voltage source 320 ₂ via signal paths 324 ₃and 324 ₄ respectively, wherein source 320 ₂ has a control inputconnected to computer 310 via signal path 322 ₂. Voltage source 320 ₂ ispreferably a DC voltage supply controllable by computer 310 to therebyimpress a DC voltage between rod pairs 360/362 and 364/366.

[0095] In the operation of QMF 302, the RF voltages applied to rods360-366 alternately attract ions to rod pairs 360/362 and 364/366,wherein this attraction increases with decreasing ion mass-to-chargeratio (m/z). Below some threshold m/z value (i.e., lighter ions), theions come into contact with one of the rods 360-366 and are accordinglyneutralized or ejected. The m/z value below which ions are neutralizedis determined by the strength and frequency of the RF signal as is knownin the art. The DC voltage applied to rods 360-366 similarly attractsions thereto wherein this attraction increases with increasing m/zvalues. Above some threshold m/z value (i.e., heavier ions), the ionscome into contact with one of the rods 360-366 and are accordinglyneutralized. The m/z value above which ions are neutralized isdetermined by the strength of the DC signal as is known in the art.Referring to FIG. 12, a plot 370 of ion intensity at the ion outlet ofQMF 302 is shown demonstrating that the RF and DC voltages applied torods 360-366 result in passage through QMF 302 only of ions having m/zvalues above a minimum m/z value m/z₁ and below a maximum m/z valuem/z₂. QMF 302 thus acts as a bandpass filter wherein the pass band ofm/z values is controlled via computer 310 by controlling the operatingstrength and frequency of the RF voltage supply 320 ₁ and by controllingthe operating strength of the DC voltage supply 320 ₂. In accordancewith an important aspect of the present invention, computer 310 isoperable, under certain operating conditions, to control the m/z valuesof ions being passed from IMS 34 to the collision cell 304 as will bedescried in greater detail hereinafter.

[0096] The collision cell 304 is of known construction, and the fillingand purging of buffer gas therein/therefrom is preferably controlled bycomputer 310 in a known manner. Alternatively, the filling and purgingof cell 304 may be manually controlled via known means. In either case,when cell 304 is filled with buffer gas, ions provided thereto by QMF302 undergo collisions with the buffer gas and fragmentation of parentions into a number of daughter ions results as is known in the art. In apreferred embodiment, the internal structure of the collision cell 304is similar to that of the quadrupole mass filter illustrated in FIG. 11except that collision cell 304 includes eight rods (poles) rather thanfour, and is accordingly referred to as an octopole collision cell. Atleast one of the voltage sources 326 ₁-326 _(L) is preferably a RFvoltage source connected between two pairs of four opposing poles,wherein computer 310 is operable to control the RF voltage source tothereby concentrate ions centrally therein and provide a low-losschannel or pipe between QMF 302 and MS 36. The buffer gas for cell 304may be, for example, Argon, Helium or Xenon, although the presentinvention contemplates using other gases provided to cell 304 via source306 or 46 as described hereinabove. The present invention contemplatesthat collision cell 304 may alternatively be configured in accordancewith any desired trapping multiple (e.g., quadrupole, hexapole, etc.).Alternatively still, collision cell 304 may me configured as anon-trapping gas collision cell. In any event, those skilled in the artwill recognize that the importance of any such collision cellarrangement lies in its ability to provide for fragmentation of enteringparent ions into daughter ions.

[0097] Referring now to FIG. 13, one preferred embodiment of a process400 for conducting sequencing analysis using the instrument 300illustrated in FIG. 9, in accordance with the present invention, isshown. Process 400 begins at step 402 where a counter variable A is setequal to an arbitrary initial number (e.g., 1). Thereafter at step 404,collision cell 304 is purged of buffer gas either manually or under thecontrol of computer 310 in a known manner. It is to be understood,however, that if no buffer gas initially exists in cell 304, step 404may be avoided. Thereafter at step 406, computer 310 is operable tocontrol QMF 302 so as to. pass ions having any m/z value therethrough.In one embodiment, computer 310 is operable to execute step 406 bydeactivating voltage sources 320 ₁ and 320 ₂ to thereby operate QMF 302in an all-pass operational mode; i.e., such that QMF 302 passes ionshaving all m/z values therethrough.

[0098] Process 400 continues from step 406 at step 408 where computer310 is operable to activate ion source 74 to thereby begin thegeneration of ions from a suitable sample source. Thereafter at step410, control computer 310 is operable to pulse ion gate 356 (FIG. 10)for a predetermined duration to thereby permit entrance of a gaseousbulk of ions from collection chamber 354 into IMS 34, and to continuallypulse the ion acceleration region of MS 36, as described hereinabove, tothereby operate MS 36 in a free running mode. Those skilled in the artwill recognize that when using embodiments of ion source 32 other thanthat shown in FIG. 10 (e.g., those of FIGS. 7A and 7B), steps 408 and410 may be combined such that computer 310 is operable to activate theion source and supply a gaseous bulk of ions to IMS 34 in a single step.In any case, process 400 continues from step 410 at step 412 where aspectrum of ion flight times (i.e., ion mass) vs. ion drift times (i.e.,ion mobilities) resulting from passage of ions through IMS 34 and MS 36,as described hereinabove, is observed.

[0099] Referring now to FIGS. 14A-14D, a graphical example of steps 410and 412 is illustrated. Signal 450 of FIG. 14A represents the voltage ation gate 356, wherein computer 310 is operable to pulse gate 356 to aninactive state for a predetermined duration at step 410 to therebypermit entrance of a bulk of gaseous ions into IMS 34. Signal 452 ofFIG. 14B represents the voltage at the ion acceleration region of TOFMS36, wherein computer 310 is operable to pulse the ion accelerationregion in a free running manner at step 410 to thereby periodicallyaccelerate ions or parts of ions toward the ion detector. A typicalvalue for the duration of deactivation of ion gate signal 450 is 100 μs,a typical value for the duration of activation of the TOFMS signal 452is 3 μs, and a typical value for the time between TOFMS signalactivation is 100 μs. However, the present invention contemplates othervalues for the foregoing signal durations, and it will be understoodthat the actual signal durations used will typically be dictated by manyfactors including sample type, analysis mode, information sought and thelike. In any case, signal 454 of FIG. 14C represents the activationstate of QMF 302, wherein computer 310 is operable throughout steps 410and 412 to maintain QMF 302 in an inactive or all-pass state; i.e. QMF302 is operable to pass ions having any m/z value therethrough. Finally,a spectrum 456 of ion drift time (corresponding to ion mobility) vs. ionflight time (corresponding to ion mass) is shown in FIG. 14Dillustrating one example of the resulting ion spectrum of step 412.

[0100] Close inspection of spectrum 456 of FIG. 14D reveals that ions a,b and g do not overlap in drift times with any other ion, while ions cand d and ions e and f overlap in their respective drift times. Ions cand d will accordingly arrive at collision cell 304 at approximately thesame time (3.5 μs), and ions e and f will accordingly arrive atcollision cell 304 at approximately the same time (4.8 μs). If collisioncell 304 was filled with buffer gas so that ion fragmentation occurred,TOFMS 36 would not be able to accurately distinguish parent and daughterions attributable to ion c from those of ion d and likewise thoseattributable to ion e from those of ion f. If, however, no such overlapsoccurred, the foregoing problem would not occur. In accordance with animportant aspect of the present invention, process 400 is configured toconduct subsequent sequencing analysis (via fragmentation) with QMF 302operating in an all-pass mode if no overlap in ion drift times areevident from step 412, but is alternatively operable to conductsubsequent sequencing analysis (via fragmentation) with QMF 302 operableto selectively filter out all but one of the ions overlapping in any onedrift time. In the latter case, the sequencing analysis is repeateduntil fragmentation spectra are produced for all ions in the originalspectrum (FIG. 14D). Thus in the example of FIG. 14D, sequencinganalysis is conducted by filling collision cell 304 with buffer gas andoperating QMF 302 to selectively filter out ions d and f, for example,such that the resulting fragmentation spectrum includes fragmentationspectra of ions a, b, c, e and g. The sequencing analysis is repeated bycontrolling QMF 302 to selectively filter out ions c and e such that theresulting fragmentation spectrum includes fragmentation spectra of atleast ions d and f. In general, the instrument 300 must be taken throughan ion generation/resulting spectrum sequence Z+1 times for any sample,wherein Z is the maximum number of ions overlapping in drift time andthe “1” accounts for the initial operation of instrument 300 in order toproduce the spectrum 456 of FIG. 14D. In the example illustrated inFIGS. 14, 15 and 16, instrument 300 must accordingly be taken throughthe ion generation/resulting spectrum sequence three times since themaximum number of ions overlapping in drift time is two (e.g., two ionsc and d overlap in drift time and, two ions f and e overlap in drifttime).

[0101] Referring again to FIG. 13, process 400 continues from step 412and step 414 where process 400 is directed to the subprocess flaggedwith the current value of A. In the first time through process 400, A=1so process 400 jumps to step 416. Thereafter at step 418, the collisioncell 304 is filled with buffer gas from buffer gas source 306 (or buffergas source 46). As with step 404, step 418 may be executed manually orunder the control of computer 310. In either case, process 420 advancesfrom step 418 to step 420 where a determination is made as to whetherthere exists any overlap in ion packet drift times. Step 420 ispreferably carried out by manually observing spectrum 456 (FIG. 14D),although the present invention contemplates that step 420 may beautomated in accordance with known techniques and therefore executed bycomputer 310. In either case, if no overlap in ion drift times arepresent in the spectrum resulting at step 412, steps 408-412 arerepeated and a spectrum of fragmented parent and daughter ions results,wherein the spectrum of fragmented parent and daughter ions may beanalyzed further for sequencing purposes. If, however, ion drift timeoverlap is observed in the first execution of step 412, process 400continues from step 420 at step 422 where QMF 302 is configured toselectively filter out desired m/z values based on the observedoverlapping drift times. Thereafter, the process counter A isincremented and steps 408-412 are repeated.

[0102] Referring now to FIGS. 15A-15D, step 422 and a second passthrough steps 408, 410 and 412 are illustrated. The ion gate signal 450and TOFMS signals 452 are identical to those shown in FIGS. 14A and 14B,but the QMF signal 458 includes an activation pulse 458 ₁ during a timeperiod encompassing the drift times of ions c and d, and an activationpulse 458 ₂ encompassing the drift times of ions e and f. It is to beunderstood that activation pulses 458 ₁ and 458 ₂ are not meant torepresent a single-signal activation of QMF 302 (i.e., “triggering”),but are instead meant to represent the activation times of QMS 302relative to known ion drift times, wherein computer 302 is operableduring each of these activation times to control the voltage sources 320₁ and 320 ₂ (FIG. 11), as described hereinabove, to thereby pass onlyions having a desired m/z value and to filter out ions having any otherm/z value. In the example spectrum illustrated in FIG. 15D, computer 310is operable to control QMF 302 during activation time 458 ₁ to pass onlyions having m/z values equal to that of ion c so that ion d iseffectively filtered out. Similarly, computer 310 is operable to controlQMF 302 during activation time 458 ₂ to pass only ions having m/z valuesequal to that of ion e so that ion f is effectively filtered out. In onepreferred embodiment of process 400, computer 310 is operable at allother times in an all-pass mode to thereby pass therethrough ions havingany m/z value. In an alternate embodiment, computer 310 may be operableto sequentially control QMF 302 during time periods corresponding to thedrift times of each of the ions, wherein computer 310 is operable duringsuch time periods to pass only ions having m/z values equal to those ofinterest. Thus, for the example spectrum 460 illustrated FIG. 15D,computer 310 may alternatively be operable to activate QMF 302 duringthe drift time of ion a to pass only ions having m/z values equal tothat of ion a, to activate QMF 302 during the drift time of ion b tothereby pass only ions having m/z values equal to that of ion b, toactivate QMF 302 during the drift time of ions c and d to pass only ionshaving m/z values equal to that of ion c, etc. In either case, thespectrum 460 of FIG. 15D results, wherein the flight times of each ofthe parent and daughter ions resulting from the fragmentation of ions a,b, c, e and g in collision cell 304 are clearly resolved. From theseflight times, the m/z values of each of the fragmented ions may bedetermined in accordance with known techniques.

[0103] Referring again to FIG. 13, process 400 advances from a secondexecution of step 412 to step 414 where process 400 is directed to aprocess section flagged by the most recent value of the countingvariable A. In this case, A=2 so process 400 is directed to step 426.Thereafter at step 428, a determination is made as to whether any ionpackets exist that have not yet been accounted for in the spectrum 460of FIG. 15D. In one preferred embodiment, step 428 is conducted manuallyvia examination of spectra 456 and 460, although the present inventioncontemplates that step 428 may alternatively be automated in a knownmanner and accordingly be executed by computer 310. In any case, if itis determined at step 428 that no ion packets are unaccounted for,process 400 advances to step 432 where process 400 is terminated. If, onthe other hand, it is determined at step 428 that there exists at leastone ion packet that has not yet been accounted for in spectrum 460,process 400 advances to step 430 where QMF 302 is configured toselectively filter out desired m/z values based on the observedoverlapping drift times. Thereafter, steps 408-412 are again repeated.

[0104] Referring now to FIGS. 16A-16D, step 430 and a third pass throughsteps 408, 410 and 412 are illustrated. The ion gate signal 450 andTOFMS signals 452 are identical to those shown in FIGS. 14A and 14B, butthe QMF signal 462 includes an activation pulse 462 ₁ during a timeperiod encompassing the drift times of ions c and d, and an activationpulse 462 ₂ encompassing the drift times of ions e and f. Again, it isto be understood that activation pulses 462 ₁ and 462 ₂ are not meant torepresent a single-signal activation of QMF 302 (i.e., “triggering”),but are instead meant to represent the activation times of QMS 302relative to known ion drift times, wherein computer 302 is operableduring each of these activation times to control the voltage sources 320₁ and 320 ₂ (FIG. 11), as described hereinabove, to thereby pass onlyions having a desired m/z value and to filter out ions having any otherm/z value. In the example spectrum illustrated in FIG. 16D, computer 310is operable to control QMF 302 during activation time 462 ₁ to pass onlyions having m/z values equal to that of ion d so that ion c iseffectively filtered out. Similarly, computer 310 is operable to controlQMF 302 during activation time 462 ₂ to pass only ions having m/z valuesequal to that of ion f so that ion e is effectively filtered out. In onepreferred embodiment of process 400, computer 310 is operable at allother times in a no-pass mode to thereby inhibit passage therethrough ofions having any m/z value. In an alternate embodiment, computer 310 maybe operable to sequentially control QMF 302 during time periodscorresponding to the drift times of each of the ions, wherein computer310 is operable during such time periods to pass only ions having m/zvalues equal to those of interest. Thus, for the example spectrum 464illustrated FIG. 16D, computer 310 may additionally be operable toactivate QMF 302 during the drift times of ions a, b and g to pass onlyions having m/z values equal to those of ions a, b and g respectively.This will result in redundant flight time information forparent/daughter ions of a, b and g, but such operation serves as anaccuracy check on the data obtained from spectrum 464. In the firstcase, the spectrum 464 of FIG. 16D results, wherein the flight times ofeach of the parent and daughter ions resulting from the fragmentation ofions d and f in collision cell 304 are clearly resolved. In the lattercase, a spectrum similar to spectrum 460 of FIG. 15D results, whereinthe flight times of each of the parent and daughter ions resulting fromthe fragmentation of ions a, b, d, f and g in collision cell 304 areclearly resolved. In either case, the m/z values of each of thefragmented ions may be determined from their associated flight times inaccordance with known techniques.

[0105] While the invention has been illustrated and described in detailin the drawings and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat all changes and modifications that come within the spirit of theinvention are desired to be protected. For example, referring to FIG.17, alternative variations of the ion mobility and mass spectrometerinstrument of FIG. 9 are illustrated, wherein ion trapping, ion massfiltering and ion fragmentation functions may, in accordance with thepresent invention, be positioned in various locations with respect tothe ion source 32, ion mobility instrument 34 and time-of-flight massspectrometer 36. In a first specific example, structure 500 represents aquadrupole mass filter, such as QMF 302 described hereinabove,structures 502 and 504 may be omitted, and structure 506 represents acollision cell such as collision cell 304. In this embodiment, ion massselection is performed prior to injecting ions into IMS 34, and ionfragmentation is performed between IMS 34 and TOFMS 36. In a secondspecific example, structure 500 represents a quadrupole mass filter,such as QMF 302 described hereinabove, structure 502 represents an iontrap, such as ion trap 152 described hereinabove, structure 504 isomitted and structure 506 represents a collision cell such as collisioncell 304 described hereinabove. In this embodiment, mass selection isperformed upon ions generated by ion source 32 and the mass selectedions are collected in the ion trap 152 prior to injection into IMS 34.Fragmentation is performed in collision cell 304 as describedhereinabove. Additionally, or alternatively, fragmentation may also beperformed in ion trap 152, as-is known in the art, if ion trap 152 issupplied with a suitable buffer gas (not shown) and/or in IMS 34 asdescribed hereinabove. In a third specific example, structure 500represents a quadrupole mass filter, such as QMF 302 describedhereinabove, structure 502 represents a collision cell such as collisioncell 304 described hereinabove, and structures 504 and 506 are omitted.In this embodiment, mass selection is performed upon ions generated byion source 32 and the mass selected ions are fragmented in collisioncell 304 prior to injection into IMS 34. Fragmentation may additionallyor alternatively be performed in IMS 34, and/or an additional collisioncell 304 may be provided as structure 506 for further fragmenting theions supplied by IMS 34. In a fourth specific example, structure 500represents a quadrupole mass filter, such as QMF 302 describedhereinabove, structure 502 represents an ion trap, such as ion trap 152described hereinabove, structure 504 represents a collision cell, suchas collision cell 304 described hereinabove, and structure 506 isomitted. In this embodiment, mass selection is performed upon ionsgenerated by ion source 32, followed by collection of the mass filteredions within ion trap 152, followed by fragmentation of the ionscollected in trap 152 either within trap 152 and/or within collisioncell 304 prior to injection of the ions into IMS 34. Furtherfragmentation may be performed within IMS 34 and/or structure 506 maydefine an additional collision cell for further ion fragmentation priorto injection of the ions into TOFMS 36. Generally, it is to beunderstood that ion mass selection and ion fragmentation may occur atvarious and multiple locations relative to ion source 32, IMS 34 andTOFMS 36. Moreover, it is to be understood that IMS 34 may be generallyconfigured as a known gas chromatograph, as illustrated hereinabove, oralternatively as a known liquid chromatograph, without detracting fromthe scope of the present invention.

[0106] Referring now to FIG. 18, another alternative embodiment 600 ofthe ion mobility and mass spectrometer instrument of the presentinvention is shown. In accordance with this aspect of the invention, amolecule separation instrument 602 serves as an ion source coupled tothe ion mobility spectrometer (IMS) instrument 34 that is, in turn,coupled to the time-of-flight mass spectrometer (TOFMS) instrument 34.Any one or more of the ion mass filtering, ion trapping and ionfragmentation functions may be interposed between the moleculeseparation unit 602 and the IMS 34 and/or between the TOFMS 36, and somespecific examples of such combinations will be described in greaterdetail hereinafter. It should be understood, however, that specificdescriptions of such combinations (as with the instrumentation shown anddescribed with respect to FIG. 17) will be described by way of exampleonly, and that other combinations of instrumentation described hereinare intended to fall within the scope of the present invention. Itshould also be understood that while FIGS. 17 and 18 are illustratedsimply as various combinations of functional blocks, actualimplementations of such combinations will typically require computercontrol of one or more of the individual components included therein viavoltage sources, one or more buffer gases, one or more vacuum pumps, andthe like as shown and described with respect to one or more of thevarious embodiments of the present invention. Such control hardware hasbeen described in detail hereinabove and has therefore been omitted fromFIGS. 17 and 18 for brevity; it being further understood that thevarious components of the instruments shown in FIGS. 17 and 18 may beoperable as described hereinabove and in any one or more of theoperational modes described therefore.

[0107] In any case, in a first specific embodiment of the instrument 600shown in FIG. 18, components 604-610 are omitted and the moleculeseparation instrument 602 may be any known instrument operable toseparate molecules over time as a function of a predefined molecularcharacteristic. With these combined instrument components, the resultinginstrument 600 is thus operable to provide additional, or at leastdifferent, molecular information in a time sequence over any of theinstruments previously described hereinabove. In this embodiment, themolecule separation instrument 602 may use any one or more of the ionsources (74, 74′, 74″, 74′″) or ion source regions (32, and includingthe gated collection chamber arrangement 354 shown in FIG. 10) forgenerating ions for separation according to the predefined molecularcharacteristic. Alternatively, instrument 602 may use any known moleculeor ion generating technique specific thereto, or may alternatively stilluse any other known molecule or ion generating technique for generatingions for separation according to the predefined molecularcharacteristic.

[0108] In one embodiment, molecule separation instrument 602 is a massspectrometer of known construction such as, for example, TOFMS 36. Inthis embodiment, ions from a suitable source are first separated in timeby instrument 602 according to ion mass/charge, then in time by IMS 34as a function of ion mobility, and then again in time by TOFMS 36 as afunction of ion mass/charge. In an alternate embodiment, moleculeseparation instrument 602 is an ion mobility instrument of knownconstruction such as, for example, IMS 34. In this embodiment, ions froma suitable source are first separated in time by IMS 34 as a function ofion mobility, and then again in time as a function of ion mobility, andthen in time as a function of ion mass/charge. In this embodiment, thetwo cascaded ion mobility instruments 602 and 34 are preferablyconfigured at least slightly differently to thereby each providecorrespondingly different ion mobility vs. time information, andexamples of a number of such different configurations will be describedin greater detail hereinafter with respect to FIG. 19.

[0109] In still another embodiment, the molecule separation instrument602 may be any known instrument or process that is operable to separatemolecules in time as a function of some dimension that is neither ionmobility nor ion mass/charge to thereby provide for additional molecularinformation over that available using any combination of the techniquesdescribed hereinabove. In other words, with the combined instrumentationjust described, molecular information may be obtained in a time sequencethat includes ion mass/charge information, ion mobility information andion information separated in time as a function of some other molecularproperty or characteristic. As one specific example, the moleculeseparation instrument 602 may be a known liquid chromatographyinstrument operable to separate ions from a suitable source over time asa function of molecule retention time (or inversely, molecule migrationrate), as is known in the art. As another example, the moleculeseparation instrument 602 may be a known gas chromatography instrument,also operable to separate ions from a suitable source over time as afunction of retention time or migration rate. Generally, the presentinvention contemplates that the molecule separation instrument 602 maybe any molecule separation instrument, including any knownchromatography instrument, operable to separate molecules (ions,specifically) over time in a dimension that is neither ion mobility norion mass/charge, and any such instrumentation is intended to fall withinthe scope of the present invention.

[0110] In another specific embodiment of the instrument 600 illustratedin FIG. 18, components 606-610 are omitted, the molecule separationinstrument 602 may be any one or combination of molecule instrumentsdescribed hereinabove, and component 604 is an ion fragmentation unitsuch as a collision cell. Component 604 may accordingly include, forexample, a collision cell such as collision cell 304 and a source ofbuffer or other ion collision promoting gas such as gas source 46 or306, all as illustrated in FIG. 9. In this embodiment, at least some ofthe ions separated in time by molecule separation instrument 602 aredirected into ion fragmentation unit 604 where they undergo collisionswith an appropriate buffer gas and fragment into daughter ions asdescribed hereinabove with respect to the description of collision cell304. At least some of the daughter ions are then directed into IMS 34for separation in time according to ion mobility, and at least some ofthe ions separated in time according to ion mobility are then directedinto TOFMS 36 for separation in time according to ion mass/charge. Withmost source samples, the inclusion of fragmentation unit 604 thusprovides for even more molecular information than that available withonly instruments 602, 34 and 36.

[0111] In yet another specific embodiment of the instrument 600illustrated in FIG. 18, components 604, 608 and 610 are omitted, themolecule separation instrument 602 may be any one or combination ofmolecule instruments described hereinabove, and component 606 is an ionmass filtering unit such as a quadrupole mass filter 302 as illustratedin FIGS. 9 and 11-12. In this embodiment, at least some of the ionsseparated in time by molecule separation instrument 602 are directedinto ion mass filter 606, wherein mass filter 606 is controlled asdescribed hereinabove with respect to the description of quadrupole massfilter 302, to allow passage therethrough only of ions having desiredmass-to-charge ratios. At least some of the ions passing through the ionmass filter 606 are then directed into IMS 34 for separation in timeaccording to ion mobility, and at least some of the ions separated intime according to ion mobility are then directed into TOFMS 36 forseparation in time according to ion mass/charge. The inclusion of ionmass filter 606 thus allows for selective analysis only of ions ofinterest; i.e., only of ions having desired mass-to-charge ratios.

[0112] In still another specific embodiment of the instrument 600illustrated in FIG. 18, components 608 and 610 are omitted, the moleculeseparation instrument 602 may be any one or combination of moleculeinstruments described hereinabove. Component 604 may be either an ionfragmentation unit, such as a collision cell arrangement as shown inFIG. 9 including collision cell 304 and buffer gas source 46 or 306, oran ion mass filtering unit such as quadrupole mass filter 302. Ifcomponent 604 is an ion fragmentation unit, then component 606 ispreferably an ion mass filtering unit such as quadrupole mass filter302. In this embodiment, at least some of the ions separated in time bymolecule separation instrument 602 are directed into ion fragmentationunit 604 where they undergo collisions with an appropriate buffer gasand fragment into daughter ions as described hereinabove with respect tothe description of collision cell 304. At least some of the daughterions are then directed into ion mass filter 606, wherein mass filter 606is controlled as described hereinabove with respect to the descriptionof quadrupole mass filter 302, to allow passage therethrough only ofdaughter ions having desired mass-to-charge ratios. At least some of theions passing through the ion mass filter 606 are then directed into IMS34 for separation in time according to ion mobility, and at least someof the ions separated in time according to ion mobility are thendirected into TOFMS 36 for separation in time according to ionmass/charge. The foregoing arrangement inclusion thus allows forselective analysis only of fragmented ions of interest; i.e., only ofions having desired mass-to-charge ratios. If, on the other hand,component 604 is an ion mass filtering unit, then component 606 ispreferably an ion fragmentation unit such as the collision cellarrangement shown in FIG. 9 including collision cell 304 and buffer gassource 46 or 306. In this embodiment, at least some of the ionsseparated in time by molecule separation instrument 602 are directedinto ion mass filtering unit 604, wherein mass filter 604 is controlledas described hereinabove with respect to the description of quadrupolemass filter 302, to allow passage therethrough only of ions havingdesired mass-to-charge ratios. At least some of ions passing through ionmass filtering unit 604 are then directed into fragmentation unit 606where they undergo collisions with an appropriate buffer gas andfragment into daughter ions as described hereinabove with respect to thedescription of collision cell 304. At least some of these fragmentedions are then directed into IMS 34 for separation in time according toion mobility, and at least some of the ions separated in time accordingto ion mobility are then directed into TOFMS 36 for separation in timeaccording to ion mass/charge. The foregoing arrangement thus allows forfragmentation and subsequent spectral analysis only of ions of interest;i.e., only of ions having desired mass-to-charge ratios.

[0113] In a further embodiment of the instrument 600 illustrated in FIG.18, components 604-608 are omitted, the molecule separation instrument602 may be any one or combination of molecule instruments describedhereinabove, and component 610 is an ion fragmentation unit such as acollision cell. Component 610 may accordingly include, for example, acollision cell such as collision cell 304 and a source of buffer orother ion collision promoting gas such as gas source 46 or 306, all asillustrated in FIG. 9. In this embodiment, at least some of the ionsseparated in time by molecule separation instrument 602 are directedinto IMS 34 for separation in time according to ion mobility, and atleast some of the ions separated in time according to ion mobility arethen directed into ion fragmentation unit 604 where they undergocollisions with an appropriate buffer gas and fragment into daughterions as described hereinabove with respect to the description ofcollision cell 304. At least some of the daughter ions are then directedinto TOFMS 36 for separation in time according to ion mass/charge. Thisarrangement provides the ability to further fragment ions that have beensequentially separated in time according to the predefined molecularcharacteristic and then according to ion mobility, prior to separationin time according to ion mass-to-charge ratio. With most source samples,the inclusion of fragmentation unit 610 thus provides for even moremolecular information than that available with only instruments 602, 34and 36.

[0114] In yet a further specific embodiment of the instrument 600illustrated in FIG. 18, components 604, 606 and 610 are omitted, themolecule separation instrument 602 may be any one or combination ofmolecule instruments described hereinabove, and component 608 is an ionmass filtering unit such as a quadrupole mass filter 302 as illustratedin FIGS. 9 and 11-12. In this embodiment, at least some of the ionsseparated in time by molecule separation instrument 602 are directedinto IMS 34 for separation in time according to ion mobility, and atleast some of the ions separated in time according to ion mobility arethen directed into ion mass filter 606, wherein mass filter 606 iscontrolled as described hereinabove with respect to the description ofquadrupole mass filter 302, to allow passage therethrough only of ionshaving desired mass-to-charge ratios. At least some of the ions passingthrough the ion mass filter 606 are then directed into TOFMS 36 forseparation in time according to ion mass/charge. The inclusion of ionmass filter 608 thus allows for selective analysis only of ions ofinterest; i.e., only of ions having desired mass-to-charge ratios.

[0115] In still a further specific embodiment of the instrument 600illustrated in FIG. 18, components 604 and 606 are omitted, the moleculeseparation instrument 602 may be any one or combination of moleculeinstruments described hereinabove. Component 608 may be either an ionfragmentation unit, such as a collision cell arrangement as shown inFIG. 9 including collision cell 304 and buffer gas source 46 or 306, oran ion mass filtering unit such as quadrupole mass filter 302. Ifcomponent 608 is an ion fragmentation unit, then component 610 ispreferably an ion mass filtering unit such as quadrupole mass filter302. In this embodiment, at least some of the ions separated in time bymolecule separation instrument 602 are directed into IMS 34 forseparation in time according to ion mobility. At least some of the ionsseparated in time according to ion mobility are then directed into ionfragmentation unit 608 where they undergo collisions with an appropriatebuffer gas and fragment into daughter ions as described hereinabove withrespect to the description of collision cell 304. At least some of thedaughter ions are then directed into ion mass filter 610, wherein massfilter 610 is controlled as described hereinabove with respect to thedescription of quadrupole mass filter 302, to allow passage therethroughonly of daughter ions having desired mass-to-charge ratios. At leastsome of the ions passing through the ion mass filter 606 are thendirected into TOFMS 36 for separation in time according to ionmass/charge. The foregoing arrangement inclusion thus allows forselective analysis only of fragmented ions of interest; i.e., only ofions having desired mass-to-charge ratios. If, on the other hand,component 608 is an ion mass filtering unit, then component 610 ispreferably an ion fragmentation unit such as the collision cellarrangement shown in FIG. 9 including collision cell 304 and buffer gassource 46 or 306. In this embodiment, at least some of the ionsseparated in time by molecule separation instrument 602 are directedinto IMS 34 for separation in time according to ion mobility. At leastsome of the ions separated in time according to ion mobility are thendirected into ion mass filtering unit 608, wherein mass filter 608 iscontrolled as described hereinabove with respect to the description ofquadrupole mass filter 302, to allow passage therethrough only of ionshaving desired mass-to-charge ratios. At least some of ions passingthrough ion mass filtering unit 608 are then directed into fragmentationunit 610 where they undergo collisions with an appropriate buffer gasand fragment into daughter ions as described hereinabove with respect tothe description of collision cell 304. At least some of ions passingthrough ion mass filtering unit 610 are then directed into TOFMS 36 forseparation in time according to ion mass/charge. The foregoingarrangement thus allows for fragmentation and subsequent spectralanalysis only of ions of interest; i.e., only of ions having desiredmass-to-charge ratios.

[0116] In still another embodiment of the instrument 600 illustrated inFIG. 18, molecule separation unit 602, as described hereinabove, IMS 34and TOFMS 36 are included, and any combination of components 604-610, aseach are described hereinabove, may also be included. Those skilled inthe art will recognize specific combinations of components 604-610 thatmay be of interest, and any such combinations are intended to fallwithin the scope of the present invention.

[0117] Referring now to FIG. 19, another preferred embodiment 700 of theion mobility and mass spectrometer instrument of the present inventionis shown. In accordance with this aspect of the present invention, twocascaded ion mobility instruments 704 (IMS #1) and 706 (IMS #2) aredisposed between an ion source 702 and a mass spectrometer 36, whereinmass spectrometer 36 may be any known mass spectrometer instrument asdescribed hereinabove. Ion source 702 may be any one, or combination of,the various ion sources 74, 74′, 74″ and 74′″ or ions source regions 32(including the ion source arrangement illustrated in FIG. 10 includingion collection chamber 354) described hereinabove. Alternatively oradditionally, ion source 702 may include a molecule separationinstrument, such as instrument 602 shown and described with respect toFIG. 18, whereby ions previously separated in time according to apredefined molecular characteristic such as ion retention time, forexample, are sequentially introduced into IMS 704. A computer 708 isincluded for controlling instrument 700, which is preferably at leaststructurally equivalent to computer 38 (FIGS. 4 and 5) or computer 310(FIG. 9), and includes a memory 710 preferably having stored thereininformation relating to the operation of instrument 700 and includingsufficient storage capacity for storing information generated byinstrument 700. Computer 708 includes an output electrically connectedto ion source 702 via a number, N, of signal paths 758, wherein N may beany positive integer, and whereby computer 708 is operable to controlion source 702 as described hereinabove with respect to any of thevarious embodiments thereof. Computer 708 further includes an inputelectrically connected to an output of an ion detector 36′ of massspectrometer 36 via signal path 756, whereby computer 708 is responsiveto an ion detection signal provided on signal path 756 by detector 36′to determine information relating to ion travel through instrument 700.

[0118] The first ion mobility instrument 704 has an ion inlet 704′coupled to an ion outlet of ion source 702, an ion outlet 704″ and anion drift tube 710 (shown in phantom) of length L1 defined therebetween,wherein drift tube 710 may be structurally equivalent to drift tube 40described with respect to IMS 34 of FIG. 4. A number, J, of outputs ofcomputer 708 are electrically connected to a corresponding number ofvoltage sources VS₁-VS_(J) via respective signal paths 712 ₁-712 _(J),wherein J may be any positive integer. Voltage sources VS₁-VS_(J) are,in turn, electrically connected to instrument 704 via respective signalpaths 714 ₁-714 _(J), whereby computer 708 is operable to control theoperation of instrument 704 via appropriate control of voltage sourcesVS₁-VS_(J) as described hereinabove. At least one such voltage source(e.g., VS₁) is electrically connected to the drift tube 710 as describedwith respect to FIG. 4, wherein computer 708 is operable to control thevoltage thereof to thereby establish and control a resultant electricfield within drift tube 710.

[0119] Drift tube 710 is also fluidly coupled to a source 716 of gas(gas #1), wherein gas #1 is preferably a known buffer gas, but mayalternatively be another gas including ambient air, and is furtherfluidly coupled to a vacuum pump 80. Gas source 716 is electricallyconnected to an output of computer 708 via signal path 718, and vacuumpump 80 is electrically connected to an output of computer 708 viasignal path 720, whereby computer 708 is operable to control the flow ofgas #1 into and out of instrument 704 as described hereinabove.

[0120] Drift tube 710 is further surrounded by a variable temperaturehousing 58 connected to a variable temperature source 60 via path 62. Anoutput of computer 708 is electrically connected to variable temperaturesource 60 via signal path 64 and is operable to control temperaturesource 60 to thereby control the temperature of the interior of drifttube 710 as described hereinabove with respect to FIG. 4.

[0121] The second ion mobility instrument 706 has an ion inlet 706′coupled to ion outlet 704″ of instrument 704, an ion outlet 706″ and anion drift tube 722 (shown in phantom) of length L2 defined therebetween,wherein drift tube 722 may be structurally equivalent to drift tube 40described with respect to IMS 34 of FIG. 4. A number, K, of outputs ofcomputer 708 are electrically connected to a corresponding number ofvoltage sources VS₁-VS_(K) via respective signal paths 724 ₁-724 _(K),wherein K may be any positive integer. Voltage sources VS₁-VS_(K) are,in turn, electrically connected to instrument 706 via respective signalpaths 726 ₁-726 _(K), whereby computer 708 is operable to control theoperation of instrument 706 via appropriate control of voltage sourcesVS₁-VS_(K) as described hereinabove. At least one such voltage source(e.g., VS₁) is electrically connected to the drift tube 722 as describedwith respect to FIG. 4, wherein computer 708 is operable to control thevoltage thereof to thereby establish and control a resultant electricfield within drift tube 722.

[0122] Drift tube 722 is also fluidly coupled to a source 728 of gas(gas #2), wherein gas #2 is preferably a known buffer gas, but mayalternatively be another gas including ambient air, and is furtherfluidly coupled to a vacuum pump 80. Gas source 728 is electricallyconnected to an output of computer 708 via signal path 730, and vacuumpump 80 is electrically connected to an output of computer 708 viasignal path 732, whereby computer 708 is operable to control the flow ofgas #2 into and out of instrument 706 as described hereinabove.

[0123] Drift tube 722 is further surrounded by a variable temperaturehousing 58 connected to a variable temperature source 60 via path 62. Anoutput of computer 708 is electrically connected to variable temperaturesource 60 via signal path 64 and is operable to control temperaturesource 60 to thereby control the temperature of the interior of drifttube 722 as described hereinabove with respect to FIG. 4.

[0124] TOFMS 36 includes a vacuum pump 130 electrically connected to anoutput of computer 708 via signal path 750, whereby computer 708 isoperable to control pump 130 to thereby establish and control a vacuumlevel within TOFMS 36. A number, M, of outputs of computer 708 areelectrically connected to a corresponding number of voltage sourcesVS₁-VS_(M) via respective signal paths 752 ₁-752 _(M), wherein M may beany positive integer. Voltage sources VS₁-VS_(M) are, in turn,electrically connected to instrument 36 via respective signal paths 754₁-754 _(M), whereby computer 708 is operable to control the operation ofinstrument 36 via appropriate control of voltage sources VS₁-VS_(M) asdescribed hereinabove. It is to be understood that while the control ofgases, temperatures, voltage sources, vacuum pumps and the like havebeen shown and described with respect to FIG. 19 as being computercontrolled, any one or more such parameters and structures mayalternatively be controlled manually.

[0125] In accordance with the present invention, ion mobilityspectrometers 704 and 706 may be configured differently from each otherto thereby provide additional or expanded molecular information overthat available with a single IMS system such as those shown in FIGS. 4,5 and 9. In one embodiment, for example, instruments 704 and 706 areconfigured such that the length L1 of instrument 704 is different fromthe length L2 of instrument 706. As a specific example of thisembodiment, L1 is preferably greater than L2 so that instruments 704,706 and 36 may be operated with a sequence of increasing sampling ratesto thereby produce three-dimensional molecular information. In thisembodiment, for example, L1 may be sized such that ion drift timetherethrough is on the order of seconds, L2 may be sized such that iondrift time therethrough is on the order of milli-seconds, and TOFMS 36may be configured such that ion flight time therethrough is on the orderof micro-seconds. Ion packets traveling through instrument 700 are thussubjected to increased sampling rates, which results inmulti-dimensional molecular information.

[0126] In an alternate embodiment of instrument 700, the variabletemperature sources 60 of the ion mobility spectrometers 704 and 706 arecontrolled such that the temperature, T1, of drift tube 710 is differentthan the temperature, T2, of drift tube 722. Generally, the collisioncross-section (collision integral), and hence ion mobility, changes atelevated temperatures more so than at lower temperatures. Thus, byoperating instruments 704 and 706 at different drift tube temperatures,ion packets traveling through instrument 700 are thus subjected to threedifferent separation criteria, which results in multi-dimensionalmolecular information. In a further embodiment, either one or both ofthe variable temperature sources 60 of ion mobility spectrometers 704and 706 may be controlled to establish a temperature gradient through acorresponding one or both of the spectrometers 704 and 706. This featureallows for an additional degree of ion separation and may also be usedwith a single ion mobility spectrometer instrument of the type describedhereinabove.

[0127] In another alternate embodiment of instrument 700, the electricfields established within drift tubes 710 and 722 are controlled, asdescribed hereinabove, such that the electric field, E1, within drifttube 710 is different from the electric field, E2, within drift tube722. At low electric fields, the ratio of electric field and buffer gasconcentration is also low, and molecular collisions with the buffer gasdoes not result in any significant temperature change. At high electricfields, however, the ratio of electric field and buffer gasconcentration is high, and molecular collisions with the buffer gasresult in the generation of heat which, as just described, changes thecollision integral. By operating instruments 704 and 706 with differentdrift tube electric fields, wherein the electric field in one of thedrift tubes is at least high enough to result in the generation of heatdue to collisions of ions with the corresponding buffer gas, ion packetstraveling through instrument 700 are thus subjected to three differentseparation criteria, which results in multi-dimensional molecularinformation. In accordance with the present invention, one of theelectric fields E1 and E2 may be a zero electric field while the otheris nonzero, or alternatively, both electric fields E1 and E2 may benon-zero fields. In a further embodiment, either one or both of theelectric fields E1 and E2 may be configured as an electric fieldgradient to thereby establish an electric field gradient through acorresponding one or both of the spectrometers 704 and 706. This featureallows for an additional degree of ion separation and may also be usedwith a single ion mobility spectrometer instrument of the type describedhereinabove.

[0128] In still another alternate embodiment of instrument 700, thegases established within drift tubes 710 and 722 are chosen such thatgas #1 within drift tube 710 is different from gas #2 within drift tube722. Generally, the collision integral is different for different buffergases, and by operating instruments 704 and 706 with different gaseswithin the respective drift tubes 710 and 722, ion packets travelingthrough instrument 700 are thus subjected to three different separationcriteria, which results in multi-dimensional molecular information. Inaccordance with the present invention, either gas #1 or gas #2 may beambient air while the other gas is a known buffer gas, or alternatively,gas #1 may be a first known buffer gas and gas #2 may be a second knownbuffer gas different from gas #1.

[0129] It is to be understood that instrument 700 may be configured withany combination of the foregoing configurations of instruments 704 and706, and all such combinations are intended to fall within the scope ofthe present invention.

What is claimed is:
 1. A method of separating ions in time, comprisingthe steps of: separating a bulk of ions in time as a function of a firstmolecular characteristic; sequentially separating in time as a functionof ion mobility at least some of said ions previously separated in timeas said function of a first molecular characteristic; and sequentiallyseparating in time as a function of ion mass at least some of said ionspreviously separated in time as said function of ion mobility.
 2. Themethod of claim 1 wherein said first molecular characteristic is ionmass-charge ratio.
 3. The method of claim 1 wherein said first molecularcharacteristic is ion mobility.
 4. The method of claim 1 wherein saidfirst molecular characteristic is ion retention time.
 5. The method ofclaim 1 further including the step of sequentially fragmenting at leastsome of said ions previously separated in time as said function of afirst molecular characteristic into daughter ions prior to the step ofsequentially separating in time as a function of ion mobility at leastsome of said ions previously separated in time as said function of afirst molecular characteristic.
 6. The method of claim 5 furtherincluding the step of selectively filtering at least some of saiddaughter ions to thereby sequentially provide daughter ions having onlydesired mass-to-charge ratios prior to the step of sequentiallyseparating in time as a function of ion mobility at least some of saidions previously separated in time as said function of a first molecularcharacteristic.
 7. The method of claim 1 further including the step ofselectively filtering at least some of said ions previously separated intime as said function of a first molecular characteristic to therebysequentially provide ions having only desired mass-to-charge ratiosprior to the step of sequentially separating in time as a function ofion mobility at least some of said ions previously separated in time assaid function of a first molecular characteristic.
 8. The method ofclaim 7 further including the step of sequentially fragmenting at leastsome of said ions having only desired mass-to-charge ratios intodaughter ions prior to the step of sequentially separating in time as afunction of ion mobility at least some of said ions previously separatedin time as said function of a first molecular characteristic.
 9. Themethod of claim 1 further including the step of sequentially fragmentingat least some of said ions previously separated in time as said functionof ion mobility into daughter ions prior to the step of sequentiallyseparating in time as a function of ion mass at least some of said ionspreviously separated in time as said function of ion mobility.
 10. Themethod of claim 9 further including the step of selectively filtering atleast some of said daughter ions to thereby sequentially providedaughter ions having only desired mass-to-charge ratios prior to thestep of sequentially separating in time as a function of ion mass atleast some of said ions previously separated in time as said function ofion mobility.
 11. The method of claim 1 further including the step ofselectively filtering at least some of said ions previously separated intime as said function of ion mobility to thereby sequentially provideions having only desired mass-to-charge ratios prior to the step ofsequentially separating in time as a function of ion mass at least someof said ions previously separated in time as said function of ionmobility.
 12. The method of claim 11 further including the step ofsequentially fragmenting at least some of said ions having only desiredmass-to-charge ratios into daughter ions prior to the step ofsequentially separating in time as a function of ion mass at least someof said ions previously separated in time as said function of ionmobility.
 13. Apparatus for separating ions in time, comprising: meansfor separating a bulk of ions in time as a function of a first molecularcharacteristic; an ion mobility spectrometer (IMS) having an ion inletcoupled to said means for separating a bulk of ions in time as afunction of a first molecular characteristic and an ion outlet, said IMSoperable to separate ions in time as a function of ion mobility; and amass spectrometer (MS) having an ion acceleration region coupled to saidion outlet of said IMS, said MS operable to separate ions in time as afunction of ion mass.
 14. The apparatus of claim 13 wherein said MSincludes an ion detector producing an ion signal as a function of ionsdetected thereat, and further including: a computer having an inputconnected to said ion detector of said MS, said computer processing saidion signal and determining therefrom information relating to at leastsome of said bulk of ions as a function of ion mass, ion mobility andsaid first molecular characteristic.
 15. The apparatus of claim 13further including an ion filtering instrument disposed between saidmeans for separating a bulk of ions in time as a function of a firstmolecular characteristic and said IMS, said ion filtering instrumentoperable to pass therethrough only ions having desired mass-to-chargeratios.
 16. The apparatus of claim 15 further including a collision celldisposed between said means for separating a bulk of ions in time as afunction of a first molecular characteristic and said IMS, saidcollision cell operable to receive a buffer gas therein whereby ionsentering said collision cell may collide with said buffer gas andfragment into daughter ions.
 17. The apparatus of claim 13 furtherincluding a collision cell disposed between said means for separating abulk of ions in time as a function of a first molecular characteristicand said IMS, said collision cell operable to receive a buffer gastherein whereby ions entering said collision cell may collide with saidbuffer gas and fragment into daughter ions.
 18. The apparatus of claim13 further including an ion filtering instrument disposed between saidIMS and said MS, said ion filtering instrument operable to passtherethrough only ions having desired mass-to-charge ratios.
 19. Theapparatus of claim 18 further including a collision cell disposedbetween said IMS and said MS, said collision cell operable to receive abuffer gas therein whereby ions entering said collision cell may collidewith said buffer gas and fragment into daughter ions.
 20. The apparatusof claim 13 further including a collision cell disposed between said IMSand said MS, said collision cell operable to receive a buffer gastherein whereby ions entering said collision cell may collide with saidbuffer gas and fragment into daughter ions.
 21. A method of separatingions in time, comprising the steps of: separating a bulk of ions in timeaccording to a first function of ion mobility; sequentially separatingin time according to a second function of ion mobility at least some ofsaid ions separated in time according to said first function of ionmobility, said second function of ion mobility different from said firstfunction of mobility; and sequentially separating in time as a functionof ion mass at least some of said ions separated in time according tosaid second function of ion mobility.
 22. The method of claim 21 furtherincluding the step of generating said bulk of ions for subsequentseparation thereof according to said first function of ion mobility. 23.The method of claim 22 wherein the step of generating said bulk of ionsincludes generating said bulk of ions via electrospray ionization. 24.The method of claim 22 wherein the step of generating said bulk of ionsincludes desorbing said bulk of ions from a surface of a sample.
 25. Themethod of claim 22 wherein the step of generating said bulk of ionsincludes the steps of: generating ions from a sample source; collectingat least some of said generated ions; and repeating the generating andcollecting steps a number of times to thereby form said bulk of ions.26. The method of claim 22 wherein the step of generating said bulk ofions includes the steps of: continually generating ions from a samplesource; and collecting a number of said continually generated ions toform said bulk of ions.
 27. The method of claim 21 wherein the step ofgenerating said bulk of ions includes separating a number of ions intime according to a predefined molecular characteristic.
 28. The methodof claim 27 wherein said predefined molecular characteristic is ionretention time.
 29. The method of claim 21 wherein said first ionmobility function corresponds to first length of ion drift and saidsecond mobility function corresponds to a second length of ion driftdifferent from said first length of ion drift.
 30. The method of claim21 wherein said first ion mobility function corresponds to separatingsaid bulk of ions at a first temperature; and wherein said second ionmobility function corresponds to separating at least some of said ionspreviously separated in time according to said first function of ionmobility at a second temperature different from said first temperature.31. The method of claim 21 wherein said first ion mobility functioncorresponds to separating said bulk of ions under the influence of afirst electric field; and wherein said second ion mobility functioncorresponds to separating at least some of said ions previouslyseparated in time according to said first function of ion mobility underthe influence of a second electric field different from said firstelectric field.
 32. The method of claim 31 wherein one of said first andsecond electric fields is a non-zero electric field and the other ofsaid first and second electric fields is a zero electric field.
 33. Themethod of claim 31 wherein said first and second electric fields areboth non-zero electric fields.
 34. The method of claim 21 wherein saidfirst ion mobility function corresponds to separating said bulk of ionsin the presence of a first gas; and wherein said second ion mobilityfunction corresponds to separating at least some of said ions previouslyseparated in time according to said first function of ion mobility inthe presence of a second gas different from said first gas.
 35. Themethod of claim 34 wherein one of said first and second gases is abuffer gas and the other of said first and second gases is ambient air.36. The method of claim 34 wherein said first gas is a first buffer gasand said second gas is a second buffer gas.
 37. Apparatus for separatingions in time, comprising: a first ion mobility spectrometer (IMS1)having an ion inlet and an ion outlet, said IMS1 operable to separateions in time according to a first function of ion mobility; a second ionmobility spectrometer (IMS2) having an ion inlet coupled to said ionoutlet of said IMS1 and an ion outlet, said IMS2 operable to separateions in time according to a second function of ion mobility differentfrom said first function of ion mobility; and a mass spectrometer havingan ion acceleration region coupled to said ion outlet of said IMS2, saidmass spectrometer operable to separate ions in time as a function of ionmass.
 38. The apparatus of claim 37 wherein said IMS1 includes a firstion drift tube defining a first length; and wherein said IMS2 includes asecond ion drift tube defining a second length different from said firstlength; and wherein said first function of ion mobility corresponds tosaid first length of said first ion drift tube and said second functionof ion mobility corresponds to said second length of said second iondrift tube.
 39. The apparatus of claim 37 wherein said IMS1 includes afirst temperature source operable to force an ion drift path of saidIMS1 to a first temperature; and wherein said IMS2 includes a secondtemperature source operable to force an ion drift path of said IMS2 to asecond temperature different from said first temperature; and whereinsaid first function of ion mobility corresponds to said firsttemperature and said second function of ion mobility corresponds to saidsecond temperature.
 40. The apparatus of claim 37 wherein said IMS1includes means for establishing a first electric field within an iondrift path of said IMS1; and wherein said IMS2 includes means forestablishing a second electric field within an ion drift path of saidIMS2, said first electric field different from said second electricfield; and wherein said first function of ion mobility corresponds tosaid first electric field and said second function-of ion mobilitycorresponds to said second electric field.
 41. The apparatus of claim 40wherein one of said first and second electric fields is a zero electricfield and the other one of said first and second electric fields is anon-zero electric field.
 42. The apparatus of claim 40 wherein saidfirst and second electric fields are both non-zero electric fields. 43.The apparatus of claim 37 wherein said IMS1 includes means forestablishing a first gas within an ion drift path of said IMS1; andwherein said IMS2 includes means for establishing a second gas within anion drift path of said IMS2, said first gas different from said secondgas; and wherein said first function of ion mobility corresponds to saidfirst gas and said second function of ion mobility corresponds to saidsecond gas.
 44. The apparatus of claim 43 wherein one of said first andsecond gases is a buffer gas and the other of said first and secondgases is ambient air.
 45. The apparatus of claim 43 wherein said firstand second gases are both buffer gases.
 46. The apparatus of claim 37further including means for generating a bulk of ions; wherein said IMS1is operable to separate said bulk of ions in time according to saidfirst function of ion mobility, said second IMS is operable to separatein time according to said second function of ion mobility at least someof said ions separated in time according to said first function of ionmobility, and said mass spectrometer is operable to separate in timeaccording to ion mass at least some of said ions previously separated intime according to said second function of ion mobility.
 47. Theapparatus of claim 46 wherein said means for generating a bulk of ionsincludes means for separating ions in time as a function of a firstmolecular characteristic prior to separation of said ions in time bysaid IMS1.
 48. The apparatus of claim 47 wherein said means forseparating ions in time as a function of a first molecularcharacteristic includes means for separating said ions in time as afunction of ion retention time.
 49. The apparatus of claim 37 furtherincluding means for electronically controlling operation of said IMS1,said IMS2 and said mass spectrometer.
 50. The apparatus of claim 37wherein said mass spectrometer includes an ion detector operable todetect arrival of ions thereat and produce an ion detection signalcorresponding thereto; and further including means for processing saidion detection signal and producing therefrom information relating toseparation of ions according to said first function of ion mobility,said second function of ion mobility and ion mass.